Process for the in vitro culture of different stages of tissue parasites

ABSTRACT

The present invention provides a method for carrying out in vitro the complete developmental sequence culture of tissular parasites, which includes culturing the parasites in a totally defined culture medium which is an axenic monophasic liquid culture medium, free of serum and free of serum-derived or cell-derived macromolecules, proteins and peptides. For obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid. For obtaining promastigote forms, the medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg liquid. Application to the in vitro culture of different stages of tissular parasites such as Leishmania,  T. cruzi,  and hamatoprotozoa is also provided.

A subject of the invention relates to a process for the in vitro cultureof different stages of the developmental cycle of a parasite. It alsorelates to the parasitic forms obtained and their biological uses.

By culture is meant, in the description which follows and the claims,both the adaptation of the parasitic form by successive passages in agiven medium, and complete differentiation when it occurs for theadaptation and the culture itself of the parasitic form.

It is known that parasites constitute a real plague causing, by theintermediary of vectors, the infection of millions of people andanimals.

Thus, leishmaniases, which are widespread throughout the world, arecaused by protozoan of the Leishmania genus which are usuallytransmitted by a sand fly, Phlebotomus. Leishmaniases of the Old Worldand those of the New World are usually distinguished according to theirgeographical localization. They have very diverse clinical forms whichdiffer significantly by their seriousness and their effect on health.Cutaneous, mucocutaneous (attacking nasal, buccal mucous and those ofthe ears) and visceral leishmaniases are distinguished.

As another parasite having a devastating effect, there can also bementioned Trypanosoma cruzi, responsible for Chagas' disease. In SouthAmerica, it causes the infection of millions of people. Over 150 speciesof wild and domestic animals can be counted which can serve as hosts tothe parasite which is transmitted to man by a bug, namely a Triatoma,which feeds on blood.

The infection can pass unnoticed for several years until thetrypanosomas attack the nervous system, the heart or the digestivesystem.

The development cycle of many parasites includes various parasiticstages. That of Leishmania, for example, includes two stages havingimportant differences at the structural, morphological, biochemical,immunological and physiological level, namely

in a vector insect, a flagellated form, called promastigote, whichmultiplies by scissiparity before acquiring its infectious form, for themammalian host, also called metacyclic,

in a mammalian host, a non-flagellated stage, called amastigote, whichexclusively parasitizes mononuclear phagocyte cells.

The differentiation into amastigotes occurs after attachment andpenetration of the promastigotes into the monocytes. But only theamastigote forms persist and multiply inside the phagolysosome of themacrophages of the infected host.

In T. cruzi, this cycle includes three different parasitic stages, anepimastigote form, multiplication form of the vector insect, and theamastigote and trypomastigote forms, which are present in the infectedhost.

At present, most of the research carried out on the diagnosis of theseparasitoses and the development of vaccines is conducted on thepromastigote form whose production in culture is easy and quick.

Now, the only form present in the infected host is the amastigote formwhich, by persisting throughout the infection, triggers the immuneresponse and participates in the development of the pathology. Thedanger of systematically extrapolating the experimental results obtainedwith the cultured promastigote forms within the scope ofimmuno-prophylactic, diagnostic or therapeutic studies aimed at theamastigote forms will be well understood.

In order to respond to this problem, various authors have beeninterested in obtaining amastigote forms. Thus the obtaining ofamastigotes from tissues of experimentally infected animals or fromcultures of infected macrophages have been reported. But this involveslong and tedious isolation methods, which moreover lead to a limitednumber of parasites being obtained, which are often degenrated and whichare incapable of multiplying and of surviving for longer than 2 to 3days.

Furthermore, such amastigote forms are contaminated with cells,fragments and molecules derived from the macrophages, tissues or plasmaof the host, designated hereafter by “cellular contaminants”, limitingor making impossible the realization of many studies.

Culturing processes under axenic conditions, that is to say in theabsence of any cellular contaminant, have been proposed for some speciesof leishmanias and for T. cruzi, but they do not allow an abundant andcontinual source of amastigote forms to be made available, and do notappear to be generally applicable to a large number of species and todifferent germs.

An aim of the invention is to resolve the above disadvantages and toprovide more satisfactory experimental models by producing the desiredparasitic stages in specific culture media, of totally definedsimplified composition.

The invention relates in particular to providing a process generallyapplicable to a large number of species of a given parasite, allowinghomogeneous populations of a given parasitic stage to be produced in acontinuous manner and in unlimited quantities.

It relates more particularly to a process allowing these stages to beobtained in a form which is free from any cellular and sericcontaminant, having the characteristic of not containing anymacromolecules. By macromolecules is meant, in the description and theclaims which follow, non-dialyzable molecules with a cut-off thresholdof 3 kDa, that is to say having an apparent molecular weight of greaterthan 3 kDa (for example seric protein such as albumin).

It also relates to the in vitro production of the complete developmentcycle of parasites under axenic and aseric conditions.

According to another aspect, the invention relates to the new parasiticforms obtained, corresponding to the different stages of the parasiticcycle and, for each stage, to the different phases of their growth.

According to another aspect, the invention relates to the applicationsof the parasitic stages obtained and of the products produced orisolated from these stages, in particular in the domain of the diagnosisof parasitoses, immunoprophylaxis and screening of drug activities.

The process according to the invention, for the in vitro culture ofdifferent stages of tissular parasites, such as leishmania, T. cruzi, oralso hematoprotozoa is characterized in that it is carried out in anaxenic and aseric, liquid single-phase culture medium, free frommacromolecules (non-dialyzable at a cut-off threshold of 3 kDa) and thatfor obtaining amastigote forms, this medium is buffered at a pH of 5.5to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid,and in particular from 400 to 550 milliosmoles/kg of liquid, or that,for obtaining promastigote forms, this medium is buffered at a pH of 7to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid,and in particular from 300 to 380 milliosmoles/kg of liquid.

The pH value of these media, within the range indicated above, ensuresthat the culture conditions are strictly controlled.

In the case of the culture of amastigotes, when the pH is greater than6.5, a tendency towards the retransformation of the amastigotes intopromastigotes is in fact noted and when it is less than 5.5, a poorgrowth is observed.

According to a preferred method of the invention, for obtainingamastigote forms, a culture medium is used containing a base medium,produced essentially from:

at least one culture medium for insect cells which has added to itinorganic salts, of Hanks' salts type,

products which are sources of amino acids, such as L-glutamine and sojabean extracts,

sugars, such as D-glucose.

As soja bean extact, there can advantageously be used that marketedunder the Trade mark trypto casein soja®.

The culture medium for insect cells is advantageously the medium 199 Hmarketed by GIBCO.

Different compositions of this 199 H^(R) medium are given in the GIBCOBRL catalogue page 48, 1992 edition.

A specially preferred composition for the production of base mediacarries the reference 042-01181 on page 48 of this catalogue, 1992edition. The 199H M medium is more especially used, to which NaHCO₃ andL-glutamine are added.

This medium, to which the above compounds are added, is advantageouslybuffered, for example with a buffer such as HEPES.

A preferred composition of the base medium contains several culturemedia for insect cells.

A base medium of this type results from the addition to the 199 H mediumas marketed by Gibco, to which the compounds mentioned above have beenadded, of another medium such as the modified 199 H medium as marketedby Flow. A composition of this medium is given in the Flow Laboratoriescatalogue, 1992 under the reference 14230-54. Before adding it to theinitial preparation, this latter modified 199 H medium is subjected to athermal treatment.

The base media defined above are new and, as such, are also a subject ofthe invention. They can be stored for several months at −20° C.

In order to avoid oxidation of the parasitic stages, this base mediumhas added to it, at the time of use, anti-oxidizing agents such ashemin, which also has the advantage of constituting a source of iron andagents with a reducing effect such as reduced glutathionee. Vitamins arealso advantageously added. A suitable mixture of vitamins includesbiotin, calcium D pantothenate, choline chloride, folic acid, inositol,nicotinamide, para-aminobenzoic acid, pyridoxine hydrochloride,riboflavin, thiamine hydrochloride, vitamin B12, some of which are usedand advantageously all of which are used.

The resultant culture media are advantageously characterized in thatthey are free from nucleotides as additives. They can be stored at +4°C. for about two weeks without alteration to their properties.

The use of the resultant culture media, as illustrated by the examplesgiven hereafter, allows amastigote forms to be produced which arecapable of mass multiplication in vitro, in a continuous manner.

The axenic and aseric media have the advantage of being free from allmacromolecules and in particular from those present in foetal calf serumand/or originating from the host cells, which can mask otherconstituents (seric and cellular proteins).

These media are particularly suitable for the amastigote forms oftissular protozoa, such as cutaneous or muco-cutaneous or visceralleishmanias, or various clones of T. cruzi, or also Plasmodium orBabesia.

As species of leishmanias, there can be mentioned L. mexicana, L.amazonensis, and L. braziliensis or also L. major, L. guyanensis and L.panamensis. Other preferred culture media, particularly suitable for theculture of the amastigote forms of visceral leishmanias, such as L.donovani, L. infantun and L. chagasi, contain, in addition, sulphurouscompounds. These are in particular sulphurated amino acids, such ascysteine, and notably the L form, and/or nutritive products such asbathocuproine sulphonic acid.

For the differentiation of promastigote forms into amastigote forms, inthe case of Leishmania, the cellular culture medium represents, relativeto the final medium, about 8 to 15% (V/V), notably of the order of 10%,the amino acids, or products which are sources of these amino acids,such as soja trypto casein ^(R) and L-glutamine, are present at a rateof about 4 to 8% (W/V), notably about 5 to 6%, the supply of sugar,notably as glucose, is advantageously carried out at a rate of about 2to 4% (W/V), notably 2 to 3%, the anti-oxidizing agents such as hemin,at a rate of 0.0002 to 0.0015% (w/V), notably of the order of 0.0005%,and glutathione at a rate of 0.01% to 0.05%, notably of the order of0.025% and the vitamin solution (100×) at a rate of 1% to 5% (V/V),notably of the order of 2%.

The sulphurous compounds, when they are used, in particular L-cysteine,are used at a rate of about 0.25 to 0.50% (W/V), notably of the order of0.3%, and bathocuproine sulphonic acid is used at a rate of about 0.004to 0.008% (W/V), notably of the order of 0.005%.

For the culture of the promastigote stages, a culture medium isadvantageously used which is produced from a medium suitable for cellculture, such as RPMI 1640 medium, to which are added amino acids suchas L-glutamine and a buffer to adjust the pH to a value of 7 to 7.5,this medium also having added to it another medium suitable for cellculture, in particular 199H M medium, containing inorganic salts such asHanks' salts, and anti-oxidizing agents, such as hemin.

This medium is therefore free from any seric contaminant and contains nomacromolecule as proved by 10% polyacrylamide gel analysis.

The 199H M medium is used at a rate of about at least 2% (V/V), notablyabout 2 to 10% and bovine hemin is used at a rate of 0.0001 to 0.0015%(W/V), notably of the order of 0.0005%.

The simplicity of preparation of such a medium from products which arealready being marketed will be observed. Moreover the absence of serumadvantageously produces an inexpensive product.

In accordance with the invention, these culture media are used in aprocess for the continuous mass production of parasitic forms.

For the adaptation and continuous culture in vitro of amastigote forms,a suitable culture medium as defined above is inoculated withpromastigotes at the end of the exponential phase, at a rate of 10⁶ to10⁷ promastigotes/ml of medium.

The conditions for carrying out the adaptation and culture areadvantageously chosen in such a way as to ensure a total transformationof the parasites in a reproducible manner.

The adaptation, then the culture, are carried out at temperatures of theorder of 28 to 36° C., and notably around 32° C. at a pH of 5.5 to 6.5.Usually a transformation of the promastigote forms into amastigote formsin excess of 90% is observed in 6 to 7 days. This transformation is forexample total in 4 days for the Leishmania, after a number of passageswhich varies according to the species studied and which generallycorresponds to 3 to 9 subcultures, and which decreases when theincubation temperature increases.

For the adaptation and continuous culture in vitro of so-called primaryculture or short-term promastigote forms of parasites, such asLeishmania, directly obtained from the amastigote forms, inoculationwith the amastigote forms as obtained above is carried out, at a rate of10⁶ to 10⁷ amastigote forms/ml of medium. Amastigote forms at the end ofthe exponential phase are advantageously used. The culture is carriedout at a temperature close to ambient temperature but preferably notexceeding 28° C. at a pH of 7 to 7.5 in a culture medium as definedabove for the development of promastigote forms.

When the use intended for these parasitic stages does not require bothaxenic and aseric conditions, without the presence of macromolecules, tobe used, it is possible within the scope of the invention to carry outthe culture in a purely axenic medium, therefore in the presence ofserum and also in the presence of macromolecules. For example RPMImedium which has foetal calf serum added to it can be used.

The short-term promastigote forms thus obtained can be used in theinoculation stage mentioned above for the purposes of thedifferentiation into amastigotes.

The transformation is very rapid and total, for example for Leishmania,in 4 days, after 2 to 5 subcultures, according to the species.

The implementation of the provisions of the invention allowsstandardized and reproducible cultures of the various formscorresponding to the various parasitic stages to be obtained, which arefree from any cellular contaminant and from macromolecules and capableof multiplying in vitro.

The provisions which precede have been described more particularlyrelative to the promastigote forms and the amastigote forms ofLeishmania whether cutaneous, muco- cutaneous or also visceralleishmanias, but also apply to the parasitic stages of T. cruzi or otherhematoprotozoa such as Plasmodium and Babesia.

These cultures can be kept for several months, even several years formany species, in particular for Leishmania, whether they are cutaneous,mucocutaneous or visceral, or for T. cruzi.

The parasitic stages are capable of undergoing long-term cultivation,that is to say over more than 50 passages in in vitro cultures, andshort-term cultivation, that is to say recently transformed frompromastigote forms or from amastigote forms (earlier form of the cycle)of less than 10 passages.

The invention also supplies the means for producing a complete parasiticcycle in vitro. It will advantageously be noted that this complete cyclecan be carried out in less than 15 days.

An embodiment of a process, according to the invention, for theproduction of stages corresponding to the developmental cycle of aparasite such as Leishmania is characterized in that it is used underthe axenic and aseric conditions defined above, in the absence ofmacromolecules and in that it comprises:.

inoculation of a suitable culture medium, as defined above, withshort-term or long-term promastigote forms, so as to obtain thedifferentiation into amastigote forms,

recovery of the amastigote forms produced, their inoculation and theirculture as indicated above so as to obtain the differentiation intopromastigote forms,

the cycle being repeated in its entirety, or partially, if desired, andindefinitely.

According to another embodiment, the short-term forms are cultivatedunder purely axenic conditions, or axenic and aseric conditions in thepresence of macromolecules.

Via the process of the invention, it is therefore possible to obtain invitro the various parasitic stages much more quickly and easily than bythe in vivo techniques currently used which involve experimentalinfections which are sometimes difficult to bring about. Theseamastigote or promastigote parasitic forms are free from any cellularcontaminant and are capable of multiplying in vitro. Thus means areavailable which allow the abundant, and even unlimited, obtaining of theparasitic stages recently, or not, differentiated from the earlierstage, in particular of Leishmania and those of T. cruzi (epimastigotes,metacyclic and bloodstream trypomastigotes and amastigotes).

Also a subject of the invention is the parasitic forms of thedevelopmental cycle of tissular protozoan, such as Leishmania or T.cruzi, as obtained by implementing the culture process defined above.

These forms are characterized in that

they are free from cellular contaminants, in particular tissularmacrophage and plasmatic contaminants accompanying the intracellularparasitic forms isolated from cell cultures or from tissues ofexperimentally-infected animals, as well as any seric contaminant, whilebeing endowed with infective power in vitro and in vivo as observed onthe intracellular forms when these are habitually infective, and theirmorphological, biochemical and immunological characteristics,

they are presented in the form of a homogeneous population relative tothe age in culture and the state of differentiation for a given stage ofthe developmental cycle, this population, originating from astandardized culture, being capable of multiplying in vitro in acontinuous manner.

The amastigote forms are particularly preferred given that theycorrespond to the forms developed during infection in man or animals.

These amastigote forms are also characterized in that they possess anenzymatic activity, more particularly a peptidase activity which isqualitatively more complex than those of the promastigotes, andquantitatively different, more particularly at the level of thecysteine-protease activities, as set out in the examples.

The invention relates more particularly to the amastigote forms of bothanthroponotic and zoonotic, or anthropozoonotic Leishmania.

They can be the amastigote forms of cutaneous or muco- cutaneousLeishmania. Amongst these, there can be mentioned L. mexicana, L.amazonensis, L. braziliensis, L. guyanensis and L. panamensis. They canalso be the amastigotes of visceral leishmanias such as L. chagasi, L.donovani or L. infantum.

Other amastigote forms according to the invention are those of variousclones of T. cruzi.

The invention also relates to the short-term promastigote forms asdefined above.

They can be populations directly transformed from amastigote forms whichhave an infective power similar to that of promastigotes recentlyisolated from an infected host.

Each of the parasitic stages, promastigote or amastigote, has differentgrowth phases during multiplication in vitro, namely a latent phase, andexponential phase and a stationary phase which correspond to thepreparation of the division, to an intense multiplication, then to anon-division stage respectively.

The invention advantageously allows forms corresponding to one of thesephases to be obtained in a targeted manner and their properties andspecific biochemical characteristics to be studied.

These forms are characterized in that they are free from cellular andseric contaminants, as well as molecules non-dialyzable at a cut-offthreshold of 3 kDa.

They are therefore populations defined according to a well-determinedphase of their growth.

The corresponding amastigote or short-term promastigote forms areparticularly preferred.

The invention also relates to the total polypeptide extracts of theseparasitic forms as obtained by lysis of the cells and recovery of thesoluble or insoluble products. These extracts are also called totalantigenic extracts in the examples. By these expressions “totalpolypeptide extracts” or “total antigenic extracts” is meant theproducts as obtained by lysis of the parasitic forms, whether they areof protein, lipid or saccharide nature.

They can be in particular total polypeptide extracts of short-termpromastigote forms and quite particularly total polypeptide extracts ofamastigote forms at different stages of their growth in vitro.

These extracts are characterized by their peptide profile as revealed ina standard manner using SDS-PAGE polyacrylamide gel, under reducingcondition or not, or on polyacrylamide gel under non-denaturingcondition, as described in the examples for certain species ofLeishmania.

The invention also relates to the antigenic, protein, glucide or lipidfractions and determinants eluted or isolated from fractions of theseextracts.

The antigenic fractions and determinants of these extracts recognized,according to an antigen-antibody type reaction, by sera of animalsimmunized with the total polypeptide extracts or sera of natural orexperimental infections are particularly preferred with regard to theimmunological applications which are a subject of the invention, and inparticular the basic specific antigenic fractions and determinants.

Products of this type correspond to the antigens expressed on thesurface of the amastigote forms and to the somatic antigens present atthe level of the flagellar pocket or of vacuolar type as revealed byimmunofluorescence techniques.

The purified or semi-purified molecules and the solutions specificallyenriched with one or more of these molecules also come within the scopeof the invention, originating from natural lyses.

Other products which are of great interest with regard to the biologicalapplications according to the invention correspond to the excretionantigens as obtained from the culture supernatants conditioned by thepromastigote forms or by the amastigote forms cultivated under theaxenic and aseric conditions of the invention, originating from naturallyses.

The same goes for the differentiation antigens secreted duringdifferentiation according to the process of the invention of thepromastigote forms into amastigote forms and that of, the amastigoteforms into promastigote forms (during the in vitro production of thecycle).

These antigenic products are recovered from the supernatants by simpleconcentration and dialysis. These supernatants therefore constitute apre-enriched purified source of antigenic products.

The invention also relates to the immunization sera as obtained afteradministration of the antigenic extracts, fractions and moleculesdefined above according to the usual techniques, and the antibodiesrecovered from these sera.

It also relates to the infection sera obtained by the infection ofanimals with the infectious amastigote forms.

The antibody content of these sera varies according to the phase of theparasitic stage and is higher against the stationary phase of theamastigote forms.

The antibodies of the invention are characterized in that they recognizethe specific peptides of amastigote or promastigote parasitic forms byproducing an antigen-antibody type reaction.

Such antibodies include those only specifically recognizing the antigensof an amastigote or promastigote parasitic form, belonging to ahomologous species, that is to say to the same species as that used forobtaining them, the recognized form being that against which they havebeen formed.

They can be for example antibodies formed against an amastigote form ofLeishmania of a given species and which only specifically recognize theantigenic peptides of the amastigote forms of this species ofLeishmania.

The antibodies of the invention also include those which in additionhave a poor recognition of the other parasitic form of the speciesconsidered.

There can be mentioned for example the anti-amastigote antibodiesrecognizing, to a lesser degree, antigens of the promastigote forms ofthe same species.

Other antibodies also of the invention are in addition capable ofrecognizing, but to a lesser degree, the parasitic forms of aheterologous species, or of another genus such as T. cruzi. Antibodiesof this type correspond for example to anti-amastigote antibodies of aspecies of Leishmania recognizing amastigote peptides of another speciesof Leishmania.

According to another aspect, the invention relates to the monoclonalantibodies as advantageously obtained according to the standardtechniques of fusion of a cell line with the spleen or ganglionlymphocytes of an animal immunized by injection with a total peptideextract, an antigenic fraction or molecule as defined above by screeningthe supernatants of the hybridomas obtained, for example according tothe Elisa or IF technique so as to reveal the antibodies directedspecifically against a parasitic form of a species, as well as theclones of hybridomas secreting these monoclonal antibodies.

These antibodies constitute tools for selectively separating orisolating specific antigens of species or of stages from a mediumcontaining them and in particular from the total polypeptide extractsmentioned above by immuno-affinity techniques.

The reaction of the above immunosera with antigenic fractions andmolecules originating from a given phase of the development of theparasitic form allows the identification and isolation of the specificantigens of this stage.

The possibility of the mass production, due to the invention, of thecultured amastigote forms makes it possible to extract the total andmessenger RNA's and, from these, to create a cDNA library.

According to another aspect, the invention therefore relates to thetotal RNA's as recovered from parasitic cultures of amastigotes orpromastigotes, and the corresponding m-RNA's and cDNA's.

By comparison with a cDNA library of corresponding promastigote forms,specific peptides of a given parasitic stage are then revealed and theirsynthesis is proceeded with if appropriate by genetic engineering.

The obtaining, in accordance with the invention, of in vitro and in vivoinfecting extracellular amastigote forms, having morphological,biological and biochemical characteristics similar to those of theintracellular amastigote forms, opens up new and numerous applicationsin the domains of research and industry.

The parasitic forms of the invention are thus particularly useful asexperimental models for carrying out a first, screening in vitro ofproducts which can be active for them in vivo.

The screening process of the invention comprises:

putting the parasitic forms, more especially the amastigote forms ascultivated under axenic conditions and notably aseric conditions, andthe promastigote forms as cultivated in a completely defined medium, incontact with the products to be tested,

incorporation of nucleotides or amino acids labelled with a radioactivegroup, for example tritiated thymidine, in order to determine theactivity of the products to be tested, or the carrying out of viabilitytests using for example a tetrazolium salt such as MTT.

In the stage of putting in contact, the parasitic forms are used atconcentrations of 10⁶ parasite/ml and the activity of the medicaments isstudied at increasing concentrations.

The products to be tested can advantageously be labelled, for examplewith a radioactive group, to determine the action mechanisms and theflow of the drugs.

Thus the invention provides a model allowing, if desired, the comparisonof the in vitro activity of medicaments on the promastigote form and onthe amastigote form at different phases of their growth for a givenparasite, and the testing of this activity on the actual form which isfound in the host.

It allows improved characterization of the medicamentous activity byrevealing either a lytic effect (leishmanicidal or trypanocidal), or aninhibitory effect on the multiplication (leishmaniostatic ortrypanostatic) of the product.

The use of these parasitic forms as experimental models also allows thechemical resistance of the parasitoses to be studied.

In fact it is known that at present the resistance to medicamentsconstitutes a significant problem. Study of the mechanisms producingthis resistance, which can easily be achieved using the models of theinvention, is therefore of great interest.

The invention also relates to a kit for screening products which can beused for the treatment of parasitoses, more especially leishmanias orChagas' disease.

This kit comprises a support such as a multi-well plate containing theparasitic forms on which is it desired to test the activity of theproduct to be studied, some of these forms being used as controls, andreagents to determine the medicamentous activity of the product on theparasitic forms.

As indicated above, studies carried out up to now have not allowedparasitoses to be satisfactorily identified.

In man, or in animals and particularly in dogs, the diagnosis ofleishmaniasis for example is most often determined either by isolatingthe parasite and identifying it (parasitological examination), or bydetecting (specific circulating) antibodies in the serum(immunoserological tests).

The industrial culture of the amastigote forms under axenic or asericconditions and in the absence of macromolecules makes available anabundant source of useful diagnostic tools.

In this way they allow the early detection, with a high degree ofsensitivity and a high specificity, of circulating antibodies directedagainst the parasites.

The invention therefore relates to a method for the diagnosis of aparasitosis in man or animals, more especially of an infection caused byLeishmania or T. cruzi, or for their detection and identification in thevector insect, characterized in that it comprises:

putting a biological sample originating from the patient or the animalto be examined in contact with an amastigote form from an axenic andaseric culture or a promastigote form from an aseric culture as definedabove, or a total polypeptide extract of these amastigote orpromastigote forms, or one or more antigens specific for this extract,purified or semi-purified,

detection of the immunological complex.

The biological sample is more particularly a biological fluid such asblood, serum or urine.

When a purified or semi-purified, of a whole parasite antigen is used,it is immobilized on a support.

Latex beads, Elisa plates or fluorescence slides are for example used.

The reaction can be revealed directly by macroscopic agglutination inthe direct or indirect agglutination test by reacting a conjugatedantibody with fluorescein (indirect immunofluorescence technique) orwith an enzyme such as peroxidase or alkaline phosphatase (ELISA tests).

A positive reaction therefore allows the presence of antibodiescirculating in the patient or animal examined to be diagnosed.

The invention also relates to a kit for implementing a method fordiagnosing a parasitosis, as defined above, in man or in animals.

This kit is characterized in that it comprises:

the antigenic reagents in immobilized form, namely the amastigote formsfrom axenic culture or axenic and aseric culture or the promastigoteforms of defined culture, the total polypeptide extracts obtained fromthese forms or the antigens specific for these extracts with, ifappropriate,

a positive control, constituted by a serum of known titer,

a negative control, as well as

the buffers and reagents which can be used for revealing theimmunological reaction.

The detection of circulating anti-parasite antibodies in a patient or ananimal can be carried out by putting in competition with the antibody ofthe sample, a specific antibody of the antigen, in particular amonoclonal antibody. The antibody advantageously contains a label, forexample a radioactive or enzymatic group.

As a variant, the diagnostic method is based on revealing the presenceof the antigenic determinants of the parasitic forms (detection ofcirculating antigens).

In this variant, the biological sample originating from the man oranimal is put in contact with specific antibodies directed against theantigens of the parasitic form, or fragments of these antibodies.

The detection of the immunological reaction is carried out for exampleusing the same antibody but labelled.

It is useful to note that the possibility provided by the invention ofdetecting circulating antigens, that is to say those which appearrapidly in the infected patient, allows early diagnosis of the diseaseto be carried out.

Antibodies directed against the promastigote or amastigote forms areused, in particular monoclonal antibodies, these forms originating fromparasites of different species of leishmanias such as L. infantum or T.cruzi.

A corresponding diagnostic kit comprises:

an appropriate solid phase serving as support for the immunologicalreaction, such as a microtitration plate,

a preparation of antibodies according to the invention as defined above,or of fragments of these antibodies, immobilized on a support,

a positive control, constituted by a serum of known titer,

a negative control, as well as

the buffers and reagents which can be used to carry out theimmunological reaction and in particular the labelled homologousantibody.

According to another aspect, the diagnostic tools of the invention allowa differential diagnosis to be carried out between several parasitoses.

In fact both in man and in animals, for example rodents, cross reactionsbetween T. cruzi, T. rangeli (trypanosome non-pathogenic to man) andvisceral or cutaneous leishmanias are observed.

The study of the parasitic forms of the invention, of the totalpolypeptide extracts and of the specific antigens defined above hasrevealed their strong immunogenic properties.

The invention therefore also relates to their use as protective agentsvis-à-vis parasitoses, more especially leishmaniases and Chagas'disease.

The vaccine compositions of the invention are characterized in that theyare developed from amastigote forms or promastigote forms from axenicand aseric culture, in the absence of macromolecules, as defined above,at different phases of their growth, or from their constituents, incombination with a vehicle and/or an administration adjuvant.

The constituents of the amastigote or promastigote forms in questioninclude the total polypeptide extracts obtained by lysis of theseparasitic forms. They also include the antigenic fractions and thespecific protective antigens isolated from the parasitic forms, but alsofrom the culture supernatants conditioned by the parasites when they arecultivated in completely defined media.

The administration of these protection agents to man or animals allowsthem to be given an overall immunity against the parasitoses in whichthey occur in the natural infection process.

Their advantageous effect was especially revealed at the level of theimmune response to cell mediation favouring the stimulation of the Tlymphocytes secreting interleukin 2, and gamma interferon (TH₁) orinhibiting the activation of the T cells secreting interleukins 4 and 5(TH₂).

These protection agents are advantageously in lyophilized form.

In the case of total polypeptide extracts, the vaccine compositions areadministered by subcutaneous route at a rate of 100 to 1000 μg in manand 100 to 500 μg in dogs in the presence of adjuvants such asmuramyldipeptide or saponin or in the presence of cytokine such as gammainterferon.

The excretion-secretion antigens of the culture supernatants metabolizedby the amastigote forms of the invention offer an original strategy inthe development of vaccines against parasitic diseases.

Their use for producing vaccines against canine or human visceralleishmaniases (L. intantum and L. chagasi) can in particular beemphasized. In fact they advantageously correspond to the forms presentin the infected host.

The various experiments carried out have allowed their immunogenicproperties and their protective effect for man or animals to berevealed.

To prepare vaccines from the said antigens, or ectoantigens, one usesthe dialyzed concentrated supernatants of amastigote cultures, or thecultures themselves containing the parasites and the supernatants, theparasites being killed for example by thermal treatment, or extracts orthese solutions.

These products are used with adjuvants such as MDP or cytokines, such asgamma interferon.

A long-term or short-term immunization protocol can be used. Thelong-term protocol is for example carried out by injection of thevaccine preparation every three weeks, on days d=0, d=21 and d=42. For ashort-term protocol, for example, two injections are given with a twoweek interval.

After the virulence test, for animals, it is verified that thehypersensitivity immunity to cell mediation has indeed been inducedtowards the activation of the TH1 cells secreting interleukin 2 andgamma interferon.

These analyses can also be carried out for man at the end of theimmunization.

Other characteristics and advantages of the invention are reported inthe examples which follow.

In these examples, reference is made to FIGS. 1 to 13, which representrespectively:

FIGS. 1a and 1 b, the differentiation curves of the promastigote formsinto amastigote forms and growth of the parasites for L. amazonensis,

FIG. 2, the percentage of amastigote forms of L. mexicana at differentculture temperatures,

FIGS. 3a and 3 b, the culture kinetics of the amastigote forms ofvarious cutaneous and mucocutaneous Leishmania, (3 a) and of a visceralleishmania (3 b),

FIGS. 4a to 4 c, the culture,kinetics of promastigote forms of cutaneousand mucocutaneous Leishmania, of visceral Leishmania of T. cruzi,

FIG. 5a, the cytological characteristics and FIG. 5b the ultrastructuralcharacteristics of the amastigote forms of Leishmania culture,

FIGS. 6a and 6 b, the SDS-PAGE electrophoresis analyses usingpolyacrylamide gel of the total polypeptide profiles of lesion andculture amastigote forms according to the invention, and of promastigoteforms of various Leishmania,

FIGS. 6c and 6 d, the electrophoresis analyses using impression gel ofthe protease activities of these forms,

FIGS. 7a to 7 d, the analyses indicated above, but relating to visceralLeishmania,

FIGS. 8a and 8 b, the analysis of the antigenic profiles of Leishmaniaby the immunoblotting technique using sera from the homologousimmunization of a rabbit,

FIGS. 9 and 10, the differentiation kinetics of amastigote formsobtained according to the invention into promastigote forms forcutaneous and mucocutaneous Leishmania and for visceral leishmaniasrespectively,

FIGS. 11 and 12, the kinetics of incorporation of [³⁵S]-methionine bythe total proteins or of excretion-secretion of L. infantum and L.amazonensis, respectively, and

FIG. 13, the degree of resistance to pentamidine of parasitic forms ofL. mexicana.

Examples 1 to 3 relate to culture media which are axenic, or axenic andaseric, but contain macromolecules. These media are described by way ofinformation from the moment they are usable during the adaptation of aparasitic form in a given medium and are then advantageously replaced,during successive passages, by axenic and aseric media, free frommolecules non-dialyzable at a cut-off threshold of 3 kDa. According tothe application envisaged for the parasitic forms, they can be used inone or more of the stages for producing the development cycle of aparasitic form.

EXAMPLE 1 Production of an Acellular Culture Medium for Amastigote Formsof Cutaneous or Mucocutaneous Leishmania or of T. cruzi

The formulation of a culture medium supporting the growth of theamastigote forms under axenic conditions of different species ofLeishmania responsible for cutaneous and mucocutaneous leishmaniases andof different strains of T. cruzi is given in Table 1.

TABLE 1 Constituents Quantities per 1 liter Basic medium 199H^(R) medium(×10) (with Hanks' salts)* 100 ml Soja tryptocasein^(R) 5 g NaHCO₃ 0.35g L-glutamine 0.75 g HEPES 5.95 g D(+) glucose 2.50 g H₂O S.Q. 800 mladditives Bovine hemin 0.015 g Foetal calf serum 200 ml *199 H mediummarketed by Gibco BRL, ref 042-01181 in the 1994 catalogue.

This medium, called in the remainder of the examples M1, is prepared asfollows:

Soja tryptocaseine ^(R), sodium bicarbonate, L-glutamine, HEPES and D(+)glucose are added successively, in the proportions indicated in thetable, to 100 ml of 199H medium (with Hanks' salts). The volume of themedium is then adjusted to 800 ml with deionized-distilled water. Thisbasic medium is kept for several months at −20° C. At the time of itsuse, bovine hemin (0.023 mM) and foetal calf serum (200 ml) which havebeen decomplemented beforehand (56° C. for 30 minutes) are added to the800 ml of basic medium. The resultant medium M1, kept at 4° C., can beused for 3 weeks without alteration to its properties. The pH of themedium is 6.5±0.1 and the osmolarity value was determined at 443.3±2.3milliosmoles per kg of water.

It should be remembered that the Hanks' salts correspond to thefollowing composition: CaCl₂, 2H₂O (1.8 g/l), KCl (4 g/l), KH₂PO₄ (0.6g/l), MgSO₄, 7H₂O (2 g/l), NaCl (80 g/l) and NaHPO₄, 7H₂O, (0.48 g/l).The concentrations indicated correspond to the 199H 10X medium.

EXAMPLE 2 Production of an Acellular Culture Medium for Amastigote Formsof Visceral Leishmania

L-cysteine (3 mM) and bathocuproine disulphonic acid (0.01 mM) are addedto the M1 medium described above. The composition of the mediumobtained, called hereafter medium M2, is given in the following Table 2:

TABLE 2 Constituents Quantities M1 medium 1000 ml L-cysteine 3 mMBathocuproine disulphonic acid 0.01 mM

EXAMPLE 3 Production of Axenic and Aseric Culture Media for AmastigoteForms of Leishmanias

Completely defined media supporting the growth of amastigote forms ofleishmanias under axenic and aseric conditions were perfected. For theamastigote forms, the foetal calf serum of medium M1 or M2 is replacedby a bovine serum albumin (BSA)—linoleic acid complex at a rate of 20 μgof linoleic acid per ml of M1 or M2 medium. The pH of the medium is6.5±0.1 and the osmolarity value was determined at 467±2.9 milliosmolesper kg of water.

The corresponding composition of the medium obtained, called M3, isgiven in Table 3 hereafter.

TABLE 3 Constituents Quantities M1 or M2 without FCS 100 ml BSA-linoleicacid 0.002% (W linoleic acid/V)

EXAMPLE 4 Production of an Cellular and Aseric Culture Medium, Free FromMacromolecules (Composed of Molecules Dialyzable at a Cut-off Thresholdof 3 kDa) for Amastigote Forms

The formulation of a medium according to the invention for thedifferentiation and culture of amastigote forms of cutaneous,mucocutaneous and visceral leishmanias and of different strains of T.cruzi is given in Table 4.

TABLE 4 Quantities for Constituents about 800 ml Basic medium 199Hmedium^(R) (×10) (with 100 ml Hanks' salts) Soja tryptocasein^(R) 5 gNaHCO₃ 0.35 g L-glutamine 0.75 g HEPES 5.95 g D(+) glucose 2.50 g H₂OS.Q. 800 ml Modified 199 H medium^(R) (×10)** 4 ml (5%) additives Bovinehemin 0.009 mM Reduced glutathione 0.08 mM Vitamin solution (×100) 2%*199H medium marketed by Gibco BRL, ref 042-01181 in the 1992 catalogue**modified 199H medium marketed by Flow Laboratories, ref 14230-54 inthe 1992 catalogue.

The medium, called in the remainder of the examples MA1, is prepared asfollows:

First of all the following basic medium is produced:

Soja tryptocasein ^(R), sodium bicarbonate, L-glutamine, HEPES and D(+)glucose are successively added in the proportions indicated in the tableto 100 ml of 199H medium (with Hanks' salts), concentrated 10 times (or10X), as marketed by Gibco BRL. The volume of the medium is thenadjusted to 800 ml with deionized-distilled water. Then modified 199Hmedium concentrated 10 times, as marketed by Flow Laboratories, isadded, which has been heated beforehand at 56° C. for 45 minutes.

This basic medium is kept for several months at −20° C. At the time ofits use, bovine hemin (0.009 mM), reduced glutathione (0.08 mM) asmarketed by Boehringer Mannheim under the reference 127744 and a vitaminsolution (2%), concentrated 100 times, such as that marketed by GibcoBRL, ref 12414-017, are added to the 800 ml of basic medium. The lattersolution contains the following vitamins with the concentrationsindicated in parentheses: biotin (0.4 mg/L), calcium D pantothenate (0.5mg/L), choline chloride (6 mg/L), folic acid (2 mg/L), i-inositol (70mg), nicotinamide (2 mg/L), paraaminobenzoic acid (2 mg/L),pyridoxine-HCl (2 mg), riboflavin (0.4 mg/L), thiamine-HCl (2 mg/L) andvitamin B12 (0.01).

The resultant MA1 medium, kept at 4° C., can be used for 3 weeks withoutalteration to its properties. The pH of the medium is 6.5±0.1 and theosmolarity value was determined at 412.3±3.1 milliosmoles per kg ofwater.

EXAMPLE 5 Production of a Culture Medium According to Example 4 Suitablefor Amastigote Forms of Visceral Leishmanias

If necessary L-cysteine (3 mM) and bathocuproine disulphonic acid (0.01mM) are added to 1000 ml of the MA1 medium of Example 4.

EXAMPLE 6 Production of Axenic and Aseric Culture Media, Free FromMacromolecules for the Promastigote Forms

The aseric medium devised for the culture of the promastigote forms isconstituted by a mixture of commercially-available culture media. 20 mlof modified 199H medium (with Hanks' salts) and 0.5 mg of bovine heminare added to one liter of RPMI 1640 medium concentrated 1.1 times andcontaining 5.95 g of Hepes. This medium is kept for 15 days at +4° C.without alteration to its properties. The pH of the medium is 7.2±0.1and the osmolarity value was determined at 353±3 milliosmoles per kg ofwater.

The formulation for one liter of culture medium supporting the growth ofthe promastigote forms of Leishmania under aseric conditions is given inTable 5 hereafter and is called MP.

TABLE 5 Constituents Quantities RPMI 1640 (1.1×) 1000 ml withL-glutamine and HEPES Modified 199H medium* (10×) 2% (W/V) Bovine hemin0.0005% (W/V) *199H medium from Gibco BRL mentioned above

EXAMPLE 7 Adaptation and Culture of Amastigote Forms of Leishmania UnderAxenic Condition

The promastigote forms of different species of Leishmania are cultured,then they are differentiated into amastigote forms.

The results of studies carried out on 19 strains of Leishmania areindicated hereafter.

Leishmania Strains

The principal characteristics (country, hosts, year of isolation andspecies) are summarized in Table 6 which follows. The subspecificcharacterization of the different species of leishmanias was carried outby genetic typing by analyzing more than 10 variable isoenzymatic loci.

TABLE 6 Number Number of of amastigotes Designation & Source Speciespassages (×10⁷/ml) MHOM/BR/79/LI-01 L. chapasi 75 7.2 MHOM/MA/67/JTMAPL. infantum 39 7.3 Clone 1 263 MHOM/MA/27/JTMAP L. infantum 48 6.9 Clone7 263 MHOM/IN(--)/61/L-13 L. donovani 64 5.9 MHOM/--/--/IT-2217 L.donovani 56 6.1 MHOM/IN/80/Ldd 8 Cl₂ L. donovani 44 5.9 MHOM/IN/80/Ldd 8Cl₂ L. donovani 41 4.8 R60 MNYC/BZ/62/M-379 L. mexicana 167 7.5MHOM/BO/83/LPZ-155 L. mexicana 97 6.2 MHOM/BR/76/LTB-012 L. amazonensis149 7.2 MHOM/BR/73/M-2269 L. amazonensis 89 5.1 MHOM/BR/75/M-2904 L.braziliensis 145 6.3 MHOM/BO/90/CS L. braziliensis 64 6.2 MHOM/BO/90/ANL. braziliensis 37 5.9 MHOM/BR/75/M-4147 L. quyanensis 94 6.6MHOM/BR/78/M-5378 L. quyanensis 42 5.4 MCHO/PA/00/M-4039 L. panamensis85 5.9 MHOM/PA/71/LS-94 L. panamensis 74 5.2 MHOM/EQ/91/A-8044 L.panamensis 39 4.8

b. Culture of Promastigote Forms

The promastigote forms of these different strains of leishmania arecultivated for the purposes of adaptation at 26±1° C. in a liquidmonophase synthetic medium, RPMI 1640, which has added to it 10% foetalcalf serum (abbreviated to FCS) which has been decomplemented beforehandat 56° C. for 30 minutes.

The composition of the medium is as follows:

RPMI 1640 10.40 g HEPES 5.95 g NaHCO₃ 2.20 g H₂O 900.00 ml

The pH is adjusted to 7.2 with 1N sodium hydroxide; the medium issterilized by filtration on a Millipore R membrane of 0.22 μm porosityand can thus be kept for one month at 4° C. At the time of its use, 100ml of decomplemented FCS is added.

The strains of leishmania are maintained under routine culture by twoweekly subcultures which consist of inoculating 5 ml of medium in a 50ml culture flask with exponential-phase promastigote forms (5×10⁵parasites per ml). The different strains are kept at −180° C. bycryo-freezing in the presence of 5% DMSO.

c. In vitro differentiation of the promastigote forms into amastigoteforms: 5×10⁷ promastigote forms, at the end of the exponential phase, ofLeishmania, obtained as indicated above, are seeded in 10 ml of M1culture medium for the promastigotes of cutaneous or mucocutaneousleishmanias and of M2 medium for those of visceral leishmanias.

On different days of the differentiation kinetics, 20 μl of parasiticsuspension is deposited on a slide, rapidly dried then fixed withabsolute methanol and stained according to the Giemsa method. Thepercentage of amastigote forms is determined by counting the number ofamastigote forms relative to the total number of parasites usingphotonic microscopy (×400) in 20 successive fields.

These cultures are carried out at a temperature comprised between 28 and36° C.

Operating as indicated above, the tests were duplicated at 28, 32, 34and 36° C. with L. mexicana and L. amazonensis and 28 and 32° C. with L.braziliensis, L. guyanensis and L. panamensis.

The various differentiation curves obtained as a function of thetemperature, during a first passage in M1 medium shows that, generally,under the conditions of these experiments,

the in vitro growth (number of parasites per ml) of the organismsstudied does not really seem to be altered by the increase in theincubation temperature of the culture. Only the multiplication kineticsof L. amazonensis at 36° C. is greatly slowed down;

the speed of differentiation (% of amastigote forms over time) as wellas the percentages of amastigote forms, determined on the eighth day ofthe culture, increase significantly and in a way which correlates withthe rise in the incubation temperature;

more than 90% of the promastigote forms are transformed into amastigoteforms by the end of the various differentiation kinetics and atincubation temperatures higher than or equal to 32° C.

In order to illustrate the results obtained, FIGS. 1a and 1 b give thenumber of parasites per ml (0) and the percentage of amastigote forms() as a function of time (in days) for L. mexicana at 34 and 36° C. Ahigh differentiation into amastigote forms is observed, which increaseswith the temperature.

Under the same conditions, but using an M2 medium, the differentiationof visceral leishmanias such as L. chagasi and L. donovani was studied.Similar percentages of amastigote forms are obtained.

It should be noted that for temperatures higher than or equal to 32° C.,the transformation of the promastigote forms into amastigote forms iscomplete in 4 days after a number of passages which varies according tothe species of leishmanias studied and which decreases when theincubation temperature is raised. At 32° C., 3 or 4, 6 to 8 and 8 or 9subcultures are necessary for cutaneous, mucocutaneous and visceralleishmaniases respectively, whilst 2 or 3 and 7 passages at 36° C. arenecessary for the cutaneous and visceral leishmaniases studied asrepresented in FIG. 2 which gives the percentage of amastigote forms asa function of the number of passages for L. mexicana at 34° C. and at36° C.

d. Growth Kinetics of the Amastigote Forms

Curves of culture kinetics for the amastigote forms of various speciesof leishmanias were established at different temperatures. FIGS. 3a and3 b show the results obtained after at least 25 subcultures of5×10⁶amastigote forms in 10 ml of M1 medium, at 32° C. for L.braziliensis (), L. guvanensis (--) and L. panamensis (-o-) and at 32°C. and 36° C. for L. chagasi.

The three characteristic phases of the cellular growth cycle usuallyobserved during the culture of the promastigote forms are found:

a latency phase (2 days),

an exponential phase during which the parasites multiply rapidly (4 to 5days according to the species of leishmanias),

a static phase which corresponds to a stage of non-division (from the6th or 7th day).

A multiplication speed is observed, calculated during the exponentialphase (corresponding to the actual doubling time) which is approximatelythe same for the various culture kinetics, but the doubling time of theamastigote forms is significantly different. It varies from 27 to 32hours according to the species studied (whereas it is only 16 to 21hours for the corresponding promastigote forms under the same inoculum,medium volume and aeration conditions). Finally, the parasiticconcentrations determined at the confluence of the cultures ofamastigote forms of leishmanias vary under the conditions of theseexperiments, from 4.8 to 7.5×10⁷ amastigote forms per ml of culture in 6or 7 days according to the species studied (see Table 5).

It is possible to cultivate the amastigote forms of the visceralleishmaniases at 34° C. and even 36° C. The growth curves are similar tothose obtained at 32° C.

EXAMPLE 8

About 10⁷ amastigote forms at the end of the exponential phase of theprincipal species of leishmanias, cultivated in M3 medium, are seeded in5 ml of MA1 medium at various temperatures. Test at 32° C. were carriedout in triplicate with L. mexicana and L. amazonensis and L. donovaniand at 36° C. for L. infantum.

If the vitamins are omitted from the medium, the amastigote forms arelyzed after the 5th subculture. In the absence of reduced glutathionethe cultures persist for 15 passages. The MA1 medium supports thecontinuous growth (greater than 40 passages) of the amastigote formsunder axenic and aseric conditions of the various species of cutaneousand mucocutaneous and visceral leishmaniases.

The culture kinetics curves of the amastigote forms of the variousspecies of leishmanias were established at different temperatures afterat least 10 subcultures of 5×10⁵ amastigote forms per ml in 5 ml of MA1medium. In a general way, they are very similar to those obtained in M3aseric medium.

Mass cultures, allowing a large production of amastigote forms, werealso carried out in 600 ml culture flasks (containing 200 to 300 ml ofuseful volume) after an intermediate passage in a 200 ml dish(containing 50 to 75 ml of useful volume). In a general way, about 10⁹parasites washed three times are obtained per 75 ml of culture.

In a general way, the amastigote forms cultivated under axenic andaseric conditions, and in the absence of macromolecules, keep theirinfectious power in vitro and in vivo. The polypeptide and proteaseprofiles are similar to those presented by the amastigote formscultivated under axenic conditions (see the results given for L.amazonensis and L. infantum).

Table 7 gives the principal characteristics of the strains of leishmaniacultivated under axenic and aseric conditions, without macromolecules,in amastigote form.

TABLE 7 average number of growth Designation and source species passages(×10⁷/ml) MHOM/MA(BE)67/IT-263 L. infantum 68 6.1 MHOM/MA/67/IT-263 L.infantum 42 6.4 clone 2 MHOM/MA/67/IT-263 L. infantum 49 7.O clone 7MHOM/../../IT-2217 L. donovani 49 7.3 MHOM/IN/80/DD8 L. donovani 26 6.5clone 2 MHYC/BZ/62/M-379 L. mexicana 72 7.6 MHOM/VE/76/JAP-78 L.amazonensis 76 6.9 MHOM/BR/76/LTB-012 L. amazonensis 42 6.9MHOM/BR/73/M-2269 L. amazonensis 85 5.9 MHOM/B6/90/CS L. braziliensis 165.8

EXAMPLE 9 Adaptation and Continuous Culture of the Promastigote Forms inCompletely Defined Media

The results of studies carried out on 25 strains of leishmania and onestrain of T. cruzi are indicated hereafter.

The principal characteristics (country, hosts, year of isolation andspecies) of these strains are summarized in Table 8 which follows. Thesubspecific characterization of the various species of leishmania and ofT. cruzi was carried out by genetic typing by analyzing more than 10variable isoenzymatic loci.

TABLE 8 Number of Number promasti- of gotes Designation and SourceSpecies passages (×10⁷/ml) MHOM/BR/79/LI-01 L. chagasi 249 6.6MHOMLMA(BE)/67/IT-263 L. infantum 89 5.6 MHOM/MA/67/IT-263 L. infantum59 6.5 clone 2 MHOM/MA/67/IT-263 L. infantum 61 7.1 clone 7 MHOM/IN/83H570 L. donovani 161 6.9 MHOM/IN/(--)/61/L-13 L. donovani 149 7.8MHOM/../../IT-2217 L. donovani 126 7.2 MHOM/IN/80/DD8 clone 2 L.donovani 129 7.3 MHYC/BZ/62/M-379 L. mexicana 229 7.9 MHOM/B6/83/LPZ-155L. mexicana 102 5.6 MHOM/VE/76/JAP-78 L. amazonensis 176 6.5MHOM/BR/76/LTB-012 L. amazonensis 142 6.9 MHOM/BR/73/M-2269 L.amazonensis 85 7.7 MHOM/BR/72/1670 L. braziliensis 111 6.5NHOM/BR/75/M-2904 L. braziliensis 99 7.1 MHOM/BO/90/CS L. braziliensis86 5.8 MHOM/BO/90/AN L. braziliensis 59 6.1 MCHO/PA/OO/M-4039 L.panamensis 107 6.6 MHOM/PA/71/LS-94 L. panamensis 136 5.3MHOM/91/EQ/A8044 L. panamensis 98 6.8 MHOM/BR/78/M-5378 L. puyanensis 765.3 MHOM/BR/75/M-4147 L. guyanensis 113 6.2 MHOM/PE/85/FR-6 L. peruviana79 5.5 schnur strain L. maior 85 5.0 LTD L. tropica 80 6.8 TEHUANTEPECSTRAIN T. cruzi 36 5.1

a. Culture of the Promastigote Forms in Completely Defined Media.

The promastigote forms of the various strains of leishmania arecultivated in a standard fashion at 26° C. in RPMI 1640 medium which hasadded to it 10% foetal calf serum (decomplemented beforehand) asindicated in Example 4, b above. The T. cruzi strain is cultivated inthe same medium, but at an incubation temperature of 28° C.

A progressive adaptation of the promastigote forms cultivated in astandard fashion in RPMI 1640 medium containing 10% foetal calf serum iscarried out in M4 medium. All the tests are carried out in duplicate at26±1° C. for the leishmania and 28±1° C. for T. cruzi in a volume of 5ml of medium in 25 cm² flasks (50 ml). The cultures are first of alldiluted to 50% in M4 medium for at least 2 passages which take placeevery 4 to 5 days, then to ⅕th, to {fraction (1/10)}th and to {fraction(1/20)}th for a number of passages which varies, according to thespecies studied, from 2 to 7 for each new concentration so as finally tocarry out subcultures corresponding to an inoculation of 5×10⁵ parasitesper ml of M4 medium.

b. Growth Kinetics of the Promastigote Forms in M4 Medium.

The culture kinetics curves of the promastigote forms of species ofleishmania and of T. cruzi are given in FIGS. 4a to 4 c. They wereestablished after at least 30 subcultures of 5×10⁶parasites in 10 ml ofM4 medium.

FIG. 4a gives the results obtained with L. braziliensis braziliensis(-□-) L. braziliensis guyanensis (--), L. braziliensis panamensis(-▪-), and L. peruviana (-o-), FIG. 4b those with L. donovani infantum(-o-) L. donovani donovani (——) and L. donovani chagasi (—▪—), FIG. 4cthose with L. mexicana mexicana (—□—), L. mexicana amazonensis (——), L.major (—▪—) and T. cruzi (—o—).

The three characteristic phases of the cellular growth cycle observed ina standard fashion during the culture of the promastigote forms arefound: a latency phase (2 days), an exponential phase (6 days) and astatic phase (from the 7th day).

The actual doubling times, calculated during the exponential phase, areapproximately the same for the various culture kinetics. They vary from23 to 30 hours according to the species of leishmania whilst theyfluctuate from 16 to 21 hours for the corresponding promastigote formscultivated in the presence of foetal calf serum under the same inoculum,medium volume and aeration conditions.

Finally, under the conditions of these experiments, the parasiticconcentrations determined at the confluence of the aseric cultures(about 7 days) of promastigote forms vary from 5.0 to 7.9×10⁷ per ml ofM4 medium. Certain species of leishmania are routinely maintained by aweekly subculture for more than 3 years.

c. Study of the Infectivity in vitro and in vivo of the PromastigoteForms of Aseric Culture.

The promastigote forms of aseric culture are harvested at 4° C. bycentrifugation at 2500 g. The parasitic pellet is then subjected tothree washings, under the same centrifugation conditions, in PBS buffer,pH 7.2. The parasitic concentration is determined using a Thoma countingchamber.

Infectivity in vitro

The results obtained with L. chagasi, L. donovani, L. amazonensis, L.mexicana and L. braziliensis are reported

According to the protocol described in Example 7,the peritonealmacrophages of Balb/C mice are infected with stationary-phasepromastigote forms at a rate of 10 parasites per macrophage.

Analysis of the kinetics of infectivity in vitro reveals that after 4hours of incubation, 24 to 70% of the macrophages according to thespecies studied have promastigote forms attached to their surface.During the kinetics, the percentages of infected macrophages increase toreach 96 to 100% after 48.hours according to the species of leishmania.

These results demonstrate that the promastigote forms obtained accordingto the invention are capable of infecting macrophages in vitro.

Infectivity in vivo

The results obtained with L. mexicana and L. amazonensis are given.

Groups of 12 Balb/C mice are respectively infected by sub-cutaneousroute in the pad of the rear right paw (the left paw serving as control)with 5×10⁷ promastigote forms (of static phase) according to theinvention of L. mexicana and of L. amazonensis. The size of the lesionwhich develops at the inoculation point is determined over time.

Characteristic lesions appear from the fifth week of infection. Theirsize reaches about 5 and 6 mm in the fourth week for L. mexicana and L.amazonensis respectively.

EXAMPLE 10 Comparison of the Extracellular Amastigote Forms According toExample 7 with Intracellular Amastigote Forms

The results obtained with L. amazonensis, L. mexicana, L. braziliensis,L. chagasi and L. donovani are reported in this example.

Obtaining the Extracellular Forms

The amastigote forms of axenic culture of the various species ofleishmania are harvested by centrifugation at 2500 g. The parasiticpellet is then subjected to three successive washings by centrifugation(same conditions) in PBS buffer, pH 7.2. For certain studies and inparticular the tests for agglutination by a lectin, a mechanicaldissociation is necessary so as to separate the naturally agglutinatedparasites. The parasitic concentration is determined using a Thomacounting chamber.

Obtaining the Intracellular Forms

The intracellular amastigote forms of L. amazonensis and of L. mexicanaare isolated from lesions developed at the inoculation point in Balb/Cmice having undergone a sub-cutaneous, injection, in the pad of the rearright paw, of 5×10⁷ stationary-phase promastigote forms. The lesions areremoved and ground up under sterile conditions in a PBS buffer, pH 7.2containing 2% glucose. A first centrifugation at a slow speed (400 g)allows the ground-up tissues to be removed. The parasitic suspension isthen washed three times with PBS by centrifugation at 2500 g. Theintracellular amastigote forms are counted in a Thoma chamber.

The intracellular amastigotes of L. chagasi and L. donovani are obtainedfrom the ground-up spleens of hamsters infected with 5×10⁷ correspondingpromastigote forms, inoculated by intraperitoneal route. These forms areisolated as described above.

Morphological Study

The cytological characteristics of the cultured amastigote formsaccording to the invention were compared with the intracellularamastigote forms by light-optical microscopy after fixation and stainingof the parasites with Giemsa stain. By way of illustration, the resultsobserved with L. amazonensis are given in FIG. 5a. On examination ofthis figure it is verified that the cultured amastigotes have generalmorphological characteristics of leishmania: well individualized nucleusand kinetoplast, but also criteria specific to the amastigote forms:round or oval forms of 2 to 5 μm on the largest axis and absence of theflagella. The arrow shown on the figure indicates amastigote forms inthe process of division.

An ultrastructural study by scanning transmission electron microscopyallowed confirmation of the ultrastructural analogy between theintracellular and cultured amastigote forms (FIG. 5b).

Study of the Infectivity in vitro of the Amastigote Forms From AxenicCulture According to the Invention

Peritoneal macrophages from Balb/C mice are recovered by washing theperitoneal cavity. They are distributed into 24-well culture plates inthe bottom of which is a 12 mm diameter glass slip, at the rate of2.5×10⁵ macrophages per well in 500 μl RPMI 10% FCS. The plates are thentransferred at 36±1° C. into an atmosphere enriched with 5% CO₂, forabout 14 hours. Washing is carried out the next day to eliminate themacrophages which have not adhered to the slip. The wells are theninfected with the amastigote forms of the exponential phase orstationary-phase at a rate of 5 parasites per macrophage.

After 4 hours of incubation at 36±1° C., all the wells are washed threetimes with RPMI medium which has added to it 10% FCS to eliminate theparasites not attached to the macrophages. Two slips are removed, washedin sterile PBS then dried rapidly, fixed and stained according to theGiemsa method, so as to evaluate the percentage of macrophages infected,and for certain species of leishmania, the number of parasites permacrophage. This operation is repeated after 24, 48 and 72 hours ofincubation. The standard deviations are calculated from two values eachcorresponding to three readings of 500 cells.

Study of the kinetics of infectivity in vitro shows that afterincubation for 4 hours, 35 to 75.4% of macrophages according to thespecies have amastigote forms attached to their surface. After 24 hours,the percentages of infected macrophages (containing amastigote forms)increase and reach 100% after 72 hours for most of the leishmania.

These results demonstrate that the amastigote forms obtained accordingto the invention are capable of attaching themselves to the macrophages,entering inside them, multiplying inside them and colonizing othermacrophages.

Comparative study of the kinetics of infection in vitro with amastigoteforms of corresponding intracellular cutaneous leishmania and visceralleishmania shows that the percentages of macrophages having amastigoteforms attached to their surface (4 hours) as well as the percentages ofmacrophages infected during the time (24, 48 and 72 hours) are verysimilar.

Study of the Infectivity in vivo

The protocols of experimental infection vary according to whethercutaneous leishmaniasis or visceral leishmania is concerned. The resultsobtained with L. mexicana and L. amazonesis on the one hand and L.chagasi and L. donovani on the other hand are reported.

Cutaneous Leishmaniasis

5×10⁶intracellular and cultured amastigote forms (of exponential orstationary-phase according to the invention) of L. mexicana and L.amazonensis, contained in 25 μl of PBS buffer, are respectively injectedinto twelve Balb/C mice by sub-cutaneous route through the right hindfoot and the left paw serving as the control. The infection kinetics aremonitored by measuring, using a precision vernier capliper, the size ofthe lesion which has developed at the inoculation point.

Characteristic lesions appear at the inoculation point between thefourth and fifth week of infection. Their size increases rapidly overtime and reaches 6 to 7 mm by the ninth week. In the mice infected by L.amazonensis, ulcerous lesions are observed from the seventh week ofinfection.

A comparative study of the experimental infection curves of the culturedamastigote forms according to the invention and of the intracellularforms shows a very close similarity.

Visceral Leishmaniasis

Groups of twelve golden hamsters are respectively infected by anintraperitoneal injection (200 μl) of 5×10⁶intracellular and culturedamastigote forms (of exponential and static phase) according to theinvention of L. chagasi and L. donovani. Every 21 days, two hamsters pergroup are sacrificed. The spleen and one kidney lobe are removed andground up under sterile conditions in 2 ml of PBS buffer. 500 μl of thesuspension is cultured in two types of culture medium (5 ml), RPMImedium with the addition of 10% FCS at 26° C. and medium M1 according toExample 1 at 32° C. Subcultures (dilution to one third) are carried outevery 4 days. The appearance of promastigote and amastigote forms ismonitored over time and the parasitic concentration is evaluated byphotonic microscopy.

The culture of the corresponding spleen homogenates on the 21st day ofinfection shows that, from the second subculture in the above RPMImedium, two to five promastigote forms of L. chagasi per field areobserved by light-optical microscopy. With L. donovani, the firstpromastigote forms are seen to appear after four subcultures. On the42nd day of the infection, 2 to 5 promastigote forms per field arevisualized from the first passage for L. chagasi and during the secondsubculture for L. donovani. On the 63rd day, more than 10 parasites perfield are counted from the first subculture for both species ofleishmania. The culture of the spleen homogenates for both types ofvisceral leishmaniasis in M1 medium at 32° C. leads to the survival,then the multiplication of the amastigote forms after 4 to 5 subculturesin this medium.

These results demonstrate, unquestionably, that the cultured amastigoteforms of the cutaneous and visceral leishmaniases as obtained accordingto the invention, are infectious in vivo. These results are confirmed byoperating under aseric conditions and in the absence of macromolecules.

Modulation of the Infectivity in vitro and in vivo of the CulturedAmastigote Forms as a Function of the Age of the Parasites Under Culture

Numerous studies provide evidence of a modification of the infectivityin vitro (vis-à-vis the peritonea macrophages) and/or in vivo (in anexperimental model) of the promastigote forms during their maturationunder culture. The sequential development from a slightly infectiousstage to a very infectious stage has been demonstrated for many speciesof leishmania.

Therefore the infectivity in vitro (vis-à-vis the peritoneal macrophagesof mice) and in vivo (in an experimental model) of the amastigote formsof axenic culture according to the invention at different phases oftheir growth was compared.

Study in vitro

The infectivity in vitro of the amastigote forms of the exponentialphase and the stationary-phase of different leishmania, as obtainedaccording to Example 4, was compared.

The amastigote forms of the exponential phase are much less infectiousthan those of the stationary-phase. From the 4th hour of incubation, themacrophages which have amastigote forms of the stationary-phase attachedto their surface are about twice as numerous. The determination of thepercentages of macrophages infected during the kinetics of infectionreveals differences of the same order in favour of the amastigote formsof the stationary-phase.

Study in vivo

Also, comparison of the kinetics of infection in vivo of the culturedamastigote forms of the exponential phase with those of thestationary-phase shows that the amastigote forms of the stationary-phaseare more virulent that those of the exponential phase.

Thus it is demonstrated that the increase in infectivity of the culturedamastigote forms of L. amazonensis, L. chagasi, and L. donovani, duringtheir growth, is accompanied by a loss of agglutination by lectin PNA(peanuts agglutinin).

EXAMPLE 11 Total Polypeptide Extracts of Cultured Amastigotes Accordingto the Invention and Comparison with Those of the IntracellularAmastigotes Protein Assay

Pellets of 10⁹ amastigote forms cultured according to the invention andintracellular amastigote forms (static phase), previously washed, arerespectively suspended in 500 μl of extraction solution (0.1% NaCl(W/V), 0.1% Triton X100 (V/V)) and subjected to a series of coldsonications of 3 10-second pulses at 30-second intervals. Thesupernatant obtained after a centrifugation at 14000 g constitutes thetotal polypeptide extract.

This extract is subjected to a protein assay according to the Bradfordmethod (BioRad Protein Assay Kit II).

Electrophoretic Analysis

SDS-PAGE electrophoretic analysis using polyacrylamide gel (non-reducingconditions) was carried out according to the protocol described byLaemmli U. K. et al., 1970, Nature, 227, 680. The proteins areconcentrated in a 5% acrylamide gel, then separated on a 10%electrophoresis gel. 50 μg of proteins are deposited per well. Proteinsof known molecular weight (Kit Low Weight, Biorad) serve as markers.Migration is carried out under 85 volts for 2 hours at 4° C. in aTris/glycine buffer, pH 8.3 containing 0.1% (W/V) of SDS.

Staining with Coomassie blue allows the polypeptide profiles to bevisualized. The free or complex polysaccharides are revealed usingSchiff's reagent.

Photographs of the gels obtained are represented in FIGS. 6a and 6 b.

In FIG. 6a, profiles 1, 2 and 3 are the total polypeptide profiles ofthe promastigote forms, of the cultured amastigote forms and of thelesion amastigote forms respectively of L. mexicana.

In FIG. 6b, profiles 1, 2 and 3 are those of the cultured amastigoteforms, of the promastigote forms and of the lesion amastigote formsrespectively of L. amazonensis.

A close similarity is observed between the total polypeptide profiles ofthe axenically-cultured amastigote forms and those of the correspondinglesion amastigote forms (profile 3).

EXAMPLE 12 Analysis of the Protease Activities in Using Gelatin-SDS PAGE

5 mg of gelatin is added to the above 10% acrylamide solution. Two typesof disclosure are then carried out: the gel is incubated for one hour atambient temperature

in a 0.01 M PBS solution, pH 7.2, containing 2.5% (V/v) Triton X100 ^(R)(to eliminate the SDS) then overnight in the PBS buffer only:

in an acetate buffer, pH 5.5, containing 2.5% (V/V) of Triton X100 ^(R),then for about 14 hours in the acetate buffer containing dithiothreitol(DTT) (49 mM).

The protease activities are revealed by staining the gel with Coomassieblue. They then appear in white on a blue background.

FIGS. 6c and 6 d represent the photographs of the impression gels. Thesefigures illustrate protease activities of cultured amastigotes (profile1), of promastigotes (profile 2) and of lesion amastigotes (profile 3),obtained from the L. mexicana and L. amazonensis strains of Example 7respectively.

As for the corresponding total polypeptide profiles, a close similarityis observed between the profiles of peptidase activities of the culturedamastigote forms and of the intracellular forms for both species ofleishmania.

EXAMPLE 13 Comparison Between Promastigote Forms and Amastigote Forms ofAxenic Culture According to the Invention Protein Concentration

The determination of the protein concentrations is carried out byspectrophotometry at 595 nm according to the Bradford method startingwith a soluble total polypeptide extract corresponding to the extractionfrom a parasitic pellet of 10⁹ parasites.

Whatever the species of leishmania studied are, the promastigote formscontain about twice as much protein as the. corresponding amastigoteforms (4.9±0.7 mg compared to 2.1±0.3 mg per ml).

Comparison of the Total Polypeptide Profiles—of Cutaneous Leishmania

An SDS-PAGE electrophoretic analysis of these extracts usingpolyacrylamide gel is carried out. The photographs of theelectrophoresis gels obtained with L. mexicana and L. amazonensis aregiven in FIGS. 6a and 6 b.

As FIG. 6a shows, comparative analysis of the polypeptide profiles ofthe promastigote forms (profile 1) and of the amastigote forms of L.mexicana (profiles 2 and 3) reveals significant quantitative andqualitative differences.

The following will be revealed:

a predominant protein with an apparent molecular weight of about 65kilodaltons (abbreviated to Kd) specifically revealed by thepromastigote forms;

three major polypeptides, having molecular weights comprised between 55and 90 Kd, revealed by the amastigote forms, but which do not appear tobe present in the total polypeptide extract of the promastigote forms.

Some differences are also revealed with L. amazonensis:

a band corresponding to a molecular weight of about 58 Kd,

several polypeptides situated in areas of molecular weight comprisedbetween 30 and 55 Kd, as well as

bands having molecular weights of greater than 90 Kd, which appear to bespecific to the amastigote forms (FIG. 6b).

Analysis of the same parasitic extracts in a polyacryl-amide gel stainedwith Schiff's reagent demonstrates that the culture, according to theinvention, of the amastigote forms is accompanied by the increasingexpression of a glycosylated molecule with a molecular weight of about130 Kd.

This molecule appears to be seldom or not at all represented in thecorresponding profiles of promastigote forms.

These promastigote forms reveal on the other hand the presence of aglycosylated molecule having a molecular weight of about 50 Kd.

Visceral Leishmanias

The total polypeptide extracts of L. donovani and L. chagasi aresubjected to an SDS-PAGE electrophoretic analysis using polyacrylamidegel.

The photographs of the total polypeptide profiles respectively obtainedare given in FIGS. 8a and 8 b.

In FIG. 7a, profiles 1 and 2 correspond to the promastigote forms and tothe cultured amastigote forms respectively of L. donovani and in FIG.7b, profiles 1 and 2 correspond to the promastigote forms and profiles 3and 4 to the cultured amastigote forms of L. chagasi.

With L. donovani (FIG. 7a) a major protein is observed revealed in theamastigote forms (profile 2) corresponding to a molecular weight ofabout 85 Kd and a band of about 105 Kd, which are not found with thepromastigote forms (profile 1), which reveal as for L. mexicana aspecific major band of about 65 Kd.

The amastigote forms of the exponential phase (profile 4) and of thestationary phase (profile 3) of L. chagasi (FIG. 7b) have two majorpolypeptides corresponding to molecular weights of about 55 and 60 Kd,which are absent from the total polypeptide profiles of thecorresponding promastigote forms of the exponential phase (profile 1)and of the static phase (profile 2).

Comparison of the Protease Activities

Comparative study of the protease activities of the correspondingamastigote and promastigote forms generally reveals profiles of activitywhich are qualitatively more complex and quantitatively greater for theamastigote forms of leishmania, allowing the appearance of proteaseactivities specific to the amastigote stage.

Of Cutaneous Leishmania

As FIG. 6c shows, the promastigote forms (profile 2) of L. mexicanaexpress a major activity with an apparent molecular weight of about 65Kd, corresponding to the surface metalloprotease, whilst the amastigoteforms (profiles 1 and 3) present:

a complex of bands which encompasses this molecular weight (from 60 to85 Kd),

activities with a higher molecular weight (greater than 110 Kd) withmigration distances varying according to the parasitic stage,

three protease activities having molecular weights comprised between 17and 35 Kd revealed specifically by the amastigote forms.

A minor band with a molecular weight of about 25 Kd, which mustcorrespond to a cysteine protease activity, only revealed for thepromastigote forms.

Such differences are also revealed for L. amazonensis (FIG. 6d). Themajor protease activities of the promastigote forms (profile 2), withmolecular weights of about 65 Kd and 115 Kd, present, for the amastigoteforms (profiles 1 and 3), significantly quicker electrophoreticmigrations. At least three protease activities having molecular weightscomprised between 24 and 36 Kd are revealed specifically by theamastigote forms; only one minor band with a molecular weight of about24 Kd and which must correspond to a cysteine protease activity isrevealed for the promastigote forms of the stationary phase.

Of Visceral Leishmanias

For L. donovani (FIG. 7c), the major protease activity of the culturedamastigote forms (profile 2) corresponding to a molecular weight ofabout 105 Kd is not expressed for the promastigote forms (profile 1).The aforementioned forms reveal a minor band of about 65 Kd, which isalso weakly found for the amastigote forms.

For L. chagasi (FIG. 7d), two protease activities, with molecularweights of about 28 and 110 Kd, are present both for the amastigoteforms of the exponential phase (profile 1) and static phase (profile 2)and for the corresponding promastigote forms (profiles 3 and 4). Thesetwo activities are however more intense for the amastigote forms of thestatic phase. The amastigote forms of the exponential and static phasepresent metalloprotease activities which are more complex than thoseexpressed by the corresponding promastigote forms. Finally, twoactivities having molecular weights of about 35 and 48 Kd are specificto the amastigote forms.

Antigenic Analysis

Comparative study of the antigenic profiles of the promastigote andamastigote forms was carried out by the immunoblotting technique usingrabbit sera immunized with total polypeptide extracts of culturedamastigote forms according to the invention.

The total polypeptide extracts of the promastigote and amastigote formsare separated on SDS-PAGE polyacrylamide gel, transferred onto anitrocellulose membrane which is incubated in the presence of thecorresponding immunization sera. The antigen/antibody complexes arerevealed by an antibody conjugated with the peroxidase directed againstthe initial antibodies.

FIG. 8a illustrates the results obtained by putting axenically-culturedamastigote forms and promastigote forms of L. amazonensis in contactwith a rabbit immunoserum directed against the amastigote forms of L.amazonensis. It is observed that four antigens are specificallyrecognized for the amastigote forms:

a major antigen with an apparent molecular weight of about 62 Kd whosemigration is slower for the promastigote forms (65 Kd),

three other molecules corresponding to respective molecular weights ofabout 81, 58 and 35 kd which are not revealed for the promastigoteforms,

two antigens of promastigote forms having molecular weights of about 45and 50 Kd, which are more readily recognized by this same immunoserum.

FIG. 8b corresponds to the amastigotes and promastigotes of L. mexicanavis-à-vis the rabbit immunoserum directed against the amastigote formsof L. mexicana. Examination of this figure shows that four antigenscorresponding to molecular weights of about 98, 90, 53 and 35 Kd arerevealed only for the amastigote forms.

Comparison of the Humoral Response vis-à-vis Axenically-culturedAmastigote Forms of the Invention and Corresponding Promastigote Forms

The intensity of the response in antibodies is evaluated by the indirectimmunofluorescence technique (IIF). The latter consists ofsemi-quantitatively detecting the circulating antibodies and has thespecific feature of using whole parasites fixed to 0.1% gutaraldehyde asantigenic substrate.

Study with Immunization Sera

Anti-L. mexicana and anti-L. amazonensis rabbit immunosera are tested indilution on the promastigote and amastigote forms of the static phase ofL. mexicana and L. amazonensis.

Rabbit sera, removed before immunization, were used to determine thepositivity thresholds of the tests. They are ½ for L. amazonensis and{fraction (1/10)} for L. mexicana.

The results are given in Table 9 which follows in which the anti-L.mexicana rabbit antiserum is called anti-Lma IS and the anti-L.amazonensis rabbit antiserum is called anti-Lma IS. The letters A and Prepresent the amastigote and promastigote forms respectively.

TABLE 9 SERA/PLATES Lma P Lma A Lmm P Lmm A anti-Lma A IS — +1/5 — +1/40anti-Lmm A IS — +1/80 — +1/40

Examination of this table shows that the two rabbit immunosera have onlypositive reactions with the cultured amastigote forms of the two speciesof leishmania. Fluroscence is observed on the surface of the amastigoteforms in the form of a line and at the level of the flagellar pocket.These results clearly demonstrate that the amastigote forms ofleishmania express, on their surface and at the level of the flagellarpocket, antigens specific to stages common to both species studied.

Study With Experimental Infection Sera

Hamster sera, infected by promastigote forms of L. mexicana and L.amazonensis respectively, are removed at different times of theexperimental infection which are 30 (D30), 50 (D50) and 221 (D221) daysfor L. amazonensis and 30 (D30), 90 (D90) and 180 (D180) days for L.mexicana. These different sera are tested in dilution on thepromastigote and amastigote forms of the static phase of L. mexicana andL. amazonensis. The positivity thresholds are determined using healthyhamster sera. It is {fraction (1/20)} whatever the species and theparasitic stage analyzed.

The results obtained are summarized in Table 10 which follows, accordingto which anti-Lma EIS and anti-Lmm EIS represent the anti-L. mexicanaand anti-L. amazonensis experimental infection sera respectively.

TABLE 10 SERA/PLATES Lma P Lma A Lmm P Lmm A anti-Lma EIS D30 — 1/40 — —anti-Lma EIS D50 1/20 1/160 — — anti-Lma EIS D221 1/20 1/160 — 1/20anti-Lmm EIS D30 — — — 1/40 anti-Lmm EIS D90 — 1/20 1/40 1/80 anti-LmmEIS D180 — 1/40 1/40 1/160

The early infection sera (D30) react specifically and only with theamastigote forms of the same species. In a general way, the antibodytiters obtained vis-à-vis the amastigote forms increase during the twoinfection kinetics. The D50 and D221 sera of L. amazonensis and D90 andD180 sera of L. mexicana also reveal slightly positive reactions({fraction (1/20)}th and {fraction (1/40)}th respectively) with thecorresponding promastigote forms. The antibody titers obtained arealways less than those revealed with the amastigote forms. Finally theD221 serum of L. amazonensis and D90, D180 sera of L. mexicana weaklyand solely recognize the amastigote forms of the heterologous species.

These results confirm the existence of antigens specific for theamastigote stage. They also demonstrate that the humoral responsegenerated during the experimental infections is more specificallydirected against epitopes expressed by the amastigote forms. Finally,they show that the antigens of amastigote forms are more capable ofdetecting an early infection.

Study of the Humoral Response During Canine Leishmaniasis

The study of the humoral response is carried out by indirectimmunofluorescence on 39 sera of dogs infected with leishmania. Itconsists of comparing the antibody response vis-à-vis two antigenicsubstrates, namely the promastigote, and amastigote forms of the staticphase of L. chagasi. Two groups of sera are used: 20 sera havingantibody titers less than or equal to {fraction (1/160)}th (“slightlypositive”) and probably corresponding to early infections and 19 serarevealing titers greater than or equal to {fraction (1/1280)}th (highlypositive), indicating well-established infections.

The positivity threshold is determined using 12 sera of dogs free fromleishmaniasis. Under the experimental conditions used with dilutions offluorescent conjugate and Evans blue, whatever the indicated antigenused, the positivity threshold is established at {fraction (1/40)}th.

The results of this study are represented in Tables 11A and 11B whichfollow in which Ldc=L. chagasi, Table 11A corresponding to the so-calledslightly positive sera and Table 11B to the so-called highly positivesera.

TABLE 11A Differences expressed in antibody titers obtained for the twoantigenic substrates Compared indicated Number Mean Minimum Maximumantigens of sera deviation deviation deviation Ldc A > LdcP 12 2.0 ± 1.21 4 Ldc A = Ldc P 3 0 0 0 Ldc A < Ldc P 5 2.2 ± 1.3 1 4

Of the 20 “slightly positive” sera, 12 of them have higher antibodytiters, 3 have equivalent titers and 5 have lower titers vis-à-vis thepromastigote forms of L. chagasi (Table 11A). The deviations expressedin higher or lower numbers of antibody titers are very great and varyfrom 1 to 4 titers.

TABLE 11B Differences expressed in antibody titers obtained for the twoantigenic substrates Compared indicated Number Mean Minimum Maximumantigens of sera deviation deviation deviation Ldc A > Ldc P 17 2.5 ±1.6 1 6 Ldc A = Ldc P 2 0 0 0 Ldc A < Ldc P 0 / / /

As Table 11B shows, the comparative analysis of the “highly positive”sera is even more convincing. In fact, all the sera reveal more intensereactions with the amastigote forms of L. chagasi, with higherdeviations in the number of antibody titers ranging from 1 to 6.

This new experimental model makes it possible to reveal an improvedsensitivity of the indirect immunofluorescence technique using asantigenic substrate the axenically-cultured amastigote forms comparedwith the corresponding promastigote forms.

EXAMPLE 14 Adaptation of the Cultured Amastigote Forms in CompletelyDefined Media

The amastigote forms of various leishmania were adapted in M3 mediumaccording to Example 3, amongst which were those of L. mexicana, L.amazonensis, L. braziliensis and L. chagasi.

A progressive adaptation in the medium is carried out as follows: about10⁷ cultured amastigote forms according to Example 4 are inoculated in 5ml of medium containing 25% then 50% then 75% and finally 100% of M4medium.

At each concentration, 4 to 5 subcultures are carried out at a rate ofone, then two passages per week.

The culture kinetics of the amastigote forms of axenic and asericculture of the different species of leishmania studied, after more than20 subcultures in M3 medium, are similar to those determined in the M1medium.

EXAMPLE 15 Production of the Parasitic Cycle in vitro

a. Under Axenic Conditions:

10⁷ amastigote forms of the exponential phase obtained according toExample 4 are seeded in 10 ml of RPMI medium with 10% FCS added to it at26° C. On the different days of the culture, 20 μl of the parasiticsuspension is deposited on a plate, dried rapidly then fixed withabsolute methanol for 2 minutes, stained according to the Giemsa methodfor 10 minutes. The percentage of promastigote forms is determined bycounting using light-optical microscopy (×400) over 20 successivefields. Successive subcultures are carried out every 4 days at a rate of10⁶parasites per ml before establishing the differentiation kineticscurves. The percentage of promastigotes obtained is represented in FIGS.9 and 10 as a function of the time (in hours) with L. braziliensis , L.guyanensis , L. panamensis (FIG. 9) and L. chagasi and L. donovani (FIG.10).

Examination of these figures shows that the transformation of thecultured amastigote forms into promastigote forms at 26° C. is completein 4 days for all the species of leishmania studied.

These promastigote forms, also called primary culture or short-termpromastigotes, seeded in the M1 medium at a rate of 10⁶parasites per ml,differentiate very rapidly (3 to 4 days) into amastigote forms at 32° C.The transformation is complete after 2 to 5 successive subculturesaccording to the species of leishmania studied.

By this process, it is therefore possible to obtain in vitro thedifferent parasitic stages of the leishmania and this can be done muchmore quickly and easily than by the in vivo techniques used at present(experimental infections). Furthermore, the parasitic forms thusproduced are free from any cellular contaminant and are capable ofmultiplying in vitro (the amastigote forms isolated from infectedtissues survive 2 to 3 days), which makes it possible to have availablean abundant source of the two parasitic stages of the principal speciesof leishmania.

The experimental infection kinetics with Balb/C mice of the short-termpromastigote forms of the exponential phase and the long-term andshort-term promastigote forms of the stationary phase were studied. Theresults obtained for L. amazonensis showed that it is possible torestore the infectivity of the “long-term” promastigote forms bycreating the parasitic cycle in vitro. The same demonstration wascarried out for visceral leishmania both in vitro and in vivo.

The infectivity in vivo of the cultured amastigote forms was alsostudied during the various successive subcultures (17, 59 and 143subcultures). The infectivity kinetics obtained are not significantlydifferent as a function of the number of passages under culture. Theseresults show that, unlike the corresponding promastigote forms, the“long-term” culture of the cultured amastigote forms does not lead to aloss of their infectivity in vivo.

b. Under Axenic and Aseric Conditions

The results obtained with L. chagasi and L. amazonensis are reported.10⁷ aserically-cultured promastigote forms at the end of the exponentialphase/beginning of the stationary phase are inoculated in 10 ml of M3medium according to the invention and incubated at 32±1° C. Thepercentage of amastigote forms obtained as a function of time isdetermined according to the protocol described previously. Thedifferentiation of the promastigote forms into amastigote forms iscomplete in 48 hours for the two species studied. It is then possible tocultivate the amastigote forms under axenic and aseric conditions asindicated in Example 10.

10⁷ cultured amastigote forms recently transformed fromaserically-cultured promastigote forms are seeded at 26±1° C. in 10 mlof M4 medium according to the invention. The transformation of theamastigote forms into promastigote forms is then complete in 3 to 5days. The promastigote forms thus obtained, also called primary-cultureor short-term promastigotes, are maintained under culture in M4 mediumby a weekly subculture as indicated in Example 5, a.

The processes for adaptation and culturing, perfected on the principalspecies of leishmania, were applied to the culture of the amastigoteforms of strains of T. cruzi, which is the agent for Chagas' disease.

The results obtained with strains SO 34 cl 1, Gamba cl 1 and SO 3 cl 1are reported hereafter, isoenzymatically typified by the analysis ofmore than 15 loci. These strains were rapidly adapted and cultivated inM1 medium at 32° C. starting from the corresponding metacyclictrypomastigote forms (infectious forms of the vector insect). Thetrypomastigote forms are obtained from the epimastigote forms(multiplication forms of the vector insect) cultivated in LIT mediumcontaining 10% FCS, according to a protocol described by Contreras etal. (1985, Mol. Biochem. Parasitol., 16, 315). 5×10⁷ epimastigote formsare incubated for 2 hours in a TAU synchronization medium (8 mMphosphate buffer, 190 mM NaCl, 17 mM KCl, 2 mM CaCl₂, pH 6), then in aTAUP differentiation medium (10 mM TAU +L-proline, 2 mM L-aspartic acid,50 mM L-glutamine and 10 mM D-glucose) until a total transformation ofthe epimastigote forms into trypomastigote forms is obtained. About5×10⁷ metacyclic trypomastigote forms are seeded in 10 ml of M1 mediumat 32° C. The differentiation into amastigote forms is complete after 4successive subcultures with strains SO 34 and Gamba and 11 subculturesfor strain S 03. Strains of this type are cultivated under axeniccondition in a continuous fashion by weekly subculture and haveundergone more than 25 passages in M1 medium.

Under light-optical microscopy, the cultured amastigotes of T. cruzipresent general morphological characteristics of the intracellularamastigote forms. A mass culture (200 ml) of strain SO 34 cl 1 wascarried out. About 10⁹ washed parasites (three washings in PBS buffer pH7.2) are obtained from 50 ml of culture.

Various tests for the differentiation of the cultured amastigote formsof strains SO34 and Gamba into epimastigote forms (multiplication formsof the vector insect) or into sanguicolous trypomastigote forms(infectious forms of the host) were also carried out. When 5×10⁶culturedamastigote forms are inoculated in 10 ml of M1 medium containing 10%calf serum, then incubated at 26° C. or at ambient temperature for 48hours, the culture contains more than 90% of epimastigote forms. On theother hand, when a culture of amastigote forms at the end of theexponential phase is incubated at 37° C. in the presence of 5% CO₂ in anM1 medium containing 30% and more of foetal calf serum for 4 days, 30 to40% of the amastigote forms are transformed into trypomastigote forms.It is therefore possible by adjusting the incubation temperatureconditions and the concentrations of foetal calf serum to produce invitro the cycle of Trypanosoma cruzi (epimastigote forms, metacyclictrypomastigote forms, amastigote forms and sanguicolous trypomastigoteforms) under axenic conditions.

EXAMPLE 16 Study of the Direct Leishmanicidal Effect on the CulturedAmastigote Forms of Nitric Oxide, a Molecule Which Brings AboutMacrophage Activation

There have recently been described antimicrobial activities of nitricoxide synthesized by macrophages activated according to a new metabolicroute leading to the synthesis by the activated macrophage ofderivatives of nitric oxide (NO, NO₂— and NO₃—) starting withL-arginine. This molecule which brings about non-specific immunity isalso responsible for anti-parasitic activities (trypanostatic effect inthe case of African trypanosomas and on Toxoplasma gondii and lyticeffect on Schistosoma mansoni and Leishmania major).

Thanks to the new experimental model of the invention, a leishmanicidalactivity directly directed against the cultured amastigote forms ofvarious species of leishmania could be demonstrated. Also, one of themechanisms leading to lysis of the amastigote forms was elucidated. Infact, the studies carried out demonstrated that the inhibition ofcis-aconitase (Krebs cycle enzyme), due to the interaction of NO withthe Fe—S prosthetic group (Iron-sulphur) of this enzyme brings aboutparasitic lysis. These results were obtained by pulsing gaseous NO (1%),carried by nitrogen (99%) free from oxygen, directly over the culturedamastigote forms of L. mexicana, L. amazonensis, L. braziliensis and L.chagasi for 15 minutes.

The culture kinetics as well as the differentiation of the amastigoteforms into promastigote forms were monitored after this treatment. Incertain experiments, media containing 100 μM of FeSO₄ (iron source) oralpha-ketoglutaric acid (3 mM) and cis-aconitate (3 mM) addedimmediately to the parasites treated with NO restore the growth of theamastigote forms.

The comparative study of different enzymatic activities, and inparticular that of the Krebs cycle, on the amastigote forms treated ornot treated with NO made it possible to reveal an inhibition ofcis-aconitase by NO.

EXAMPLE 17 Use of Leishmanial Exoantigens in an ELISA Technique for theDetection of Circulating Antibodies in Visceral Leishmaniasis

a) Protocol

Sensitization of the Plates

The supernatants of axenic and aseric cultures of promastigote forms ofL. infantum (6, 15 and 21 days) are collected by centrifugation andfiltered on a 0.22 μm Millipore ^(R) membrane. They are diluted to 50%in a carbonate buffer (0.5 M, pH 9.6) so as to obtain respectiveconcentrations of 3.5, 4.6 and 7.0 μg/ml (protein equivalent).

100 μl of these solutions is deposited in each well of themicrotitration plates. Sensitization lasts for 2 hours t 37° C., thenovernight at 4° C.

Washings

3 5-minute washings are carried out by repassage through filter paper ina washing buffer (0.01 M PBS, pH 7.2 containing 0.05% Tween 80).

Saturation of the Non-specific Fixation Sites

100 μl of fixing buffer (washing buffer containing 0.25% gelatin) isdistributed into each well, then the mixture is incubated for 30 minutesat 37° C.

Washings

3 5-minute washings in a washing buffer are carried out.

Distribution of the Sera

Dog or human sera are diluted to {fraction (1/200)}th in the fixingbuffer. 100 μl of serum is deposited per cell. Incubation is carried outfor 30 minutes at 37° C.

Washings

3.5-minute washings in a washing buffer are carried out.

Deposit of the corresponding anti-immunoglobulin marked with peroxidase.

This reagent, diluted to {fraction (1/500)}th, is deposited at a rate of100 μl per cell. The mixture is then incubated for 30 minutes at 37° C.

Washings

5 5-minute washings in a washing buffer are carried out.

Revealing the Peroxidase Activity

200 μl of reagent (1 mg ABTS in 12 ml of 50 mM citrate buffer, pH 4,with 5 μl of 30 vol. hydrogen peroxide added to it) is put in each celland the preparations are incubated for 30 minutes at ambienttemperature.

Reading

A spectrophotometer is used at 414 nm (green coloration).

b) Results

The differences obtained between the optical density averages of thenegative and positive sera (under indirect immunofluorescence) allowinfections with L. infantum to be revealed (see Table 12).

TABLE 12 Negative sera Positive sera Supernatant 6 0.128 + 0.025 0.313 +0.035 days Supernatant 15 0.080 + 0.012 0.293 + 0.020 days Supernatant21 0.062 + 0.010 0.257 = 0.039 days

EXAMPLE 18 Production of the Excretion/Secretion Antigens From theCulture Supernatants Metabolized by the Different Parasitic Stages ofLeishmania

The supernatants metabolized by the parasites of aseric culture,obtained at different times during the culture, are separated from thepromastigote or amastigote forms by centrifugation then filtration on a0.22 μm Millipore membrane treated beforehand with bacitracin (1 mg/ml).The sample is then concentrated by ultrafiltration on Mini Ultrassette(Filtron, cut-off threshold: 3 kDa), dialyzed overnight againstdistilled water and finally lyophilized. This process allows the culturesupernatants to be concentrated more than 400 times.

The protein assays are carried out by spectrophotometry at 595 nmaccording to the Bradford method (Biorad Protein Assay Kit II).According to the species of leishmania, from 1 to 4 μg antigen proteinequivalent is thus obtained per ml of non-concentrated supernatant.

Biochemical and immunological characterizations of theexcretion/secretion antigens of L. infantum and L. amazonensis:

The parasitic exoantigens are soluble molecules naturally released inthe blood of the infected hosts and in the supernatants of in vitrocultures of these parasites. A distinction must be made between the trueexoantigens (AES) resulting from the metabolism of the parasites, andthe antigens resulting from their natural or artificial lysis.

According to the invention, incorporation of methionine ³⁵S (1.85 MBq)is carried out from the third day (start of the exponential phase) ofthe culture of promastigote forms of L. amazonensis (MHOM/FE/16/JAP-78:diffuse cutaneous leishmaniasis ) and of L. infantum (MOHM/MA/67/IT-263clone 2: human visceral leishmaniasis ) inoculated at a rate of 5×10⁵parasites/ml in a synthetic medium free from macromolecules. The AES'sand the total parasitic polypeptide extracts (TPE) are analyzed usingSDS-PAGE (10% polyacrylamide), under non-reducing conditions.

The kinetics of the incorporation of [³⁵S]-methionine by the total orexcretion/secretion proteins of L. infantum and L. amazonensis arerespectively reported in FIGS. 11 and 12.

In FIG. 11, A to D correspond to the total proteins extracted 24 hrs(A), 48 hrs (B), 72 hrs (C) and 96 hrs (D) after the addition of[³⁵S]-methionine; E to H correspond to excreted/secreted proteinsremoved 24 hrs (E), 48 hrs (F), 72 hrs (G) and 96. hrs (H) after theaddition of [35S]-methionine.

In FIG. 12, A to D correspond to the total proteins extracted 24 hrs(A), 48 hrs (B), 72 hrs (C) and 96 hrs (D) after the addition of[³⁵S]-methionine; E to H correspond to the excreted/secreted proteinsremoved 24 hrs (E), 48 hrs (F), 72 hrs (G) and 96 hrs (H) after theaddition of [35S]-methionine.

The radiolabelled profiles of the AES's are much less complex than thoseof the TPE's whatever the species studied. Only 16 excretion-secretionproteins are revealed both for L. infantum and for L. amazonensis.

Immunoprecipitations (IP) were carried out:

with sera of human or canine natural infections vis-à-vis the AES's andthe TPE's (6 days of culture) of L. infantum

with sera of the experimental infestation of hamsters vis-à-vis theAES's and TPE's (6 days of culture) of L. amazonensis

with rabbit immunization sera vis-à-vis the AES's and PE's (6 days ofculture) of L. amazonensis.

The results demonstrate immunogenicity of the AES's during natural orexperimental infections. For the two parasites, certain antigens arerecognized only at the level of the TPE's whilst others are peculiar tothe AES's. For L. infantum, a similar humoral -response is observed for5 of the 6 human sera studied (2 doublets at about 70 kDa and about 45kDa) vis-à-vis the AES's and the PE's; the dog serum recognizesdifferent antigens (major antigens at about 90 and 66 kDa). For thehamster, an evolution of the humoral response exists vis-à-vis the AES'sof L. amazonensis during the experimental infection. The antigensrecognized at the time of an experimental or natural infection aredifferent from those which are immunoprecipitated by an anti-TPEimmunization serum in rabbits.

These results show that the AES's are capable of inducing an antibodyresponse during infection by L. amazonensis and L. infantum. Takingaccount of their immunogenicity, the AES's constitute very useful toolswith regard to leishmaniases in the domains of diagnostics andvaccination.

EXAMPLE 19 Production of Monoclonal Antibodies AgainstExcretion/Secretion Antigens of L. amazonensis Protocol UsedImmunization

2 μg of antigen (supernatant of aseric culture metabolized bypromastigote forms at the end of the exponential phase of L.amazonensis, concentrated 200 times and dialyzed) is injected bysub-cutaneous route into the pad of the rear paw. The antigenpreparation injected is called hereafter “Ag”.

A protocol for rapid immunization of a Balb/C mouse is carried out asfollows: D-₁₀: Ag+Freund's complete adjuvant (FCA) (V/V); D-₇ and D-₄:Ag+FIA; D₀: recovery of the cells of the popliteal ganglia draining therear paw.

Fusion

The myelomatous cells (X 63) are fused (D₀) with the lymphocyte cells ina ½ ratio in the presence of polyethylene glycol 4000 (50% PEG).

Screening

On D+₇, 442 wells (38%) had at least one cellular clone developing and66 (5.7%) had confluent cells. The supernatants of the confluent cellsare tested either by indirect immunofluorescence (IIF), or by the ELISAtest. 19 of them were revealed to be positive using IIF, 9 using ELISAand 7 in both tests. In total, more than 1000 wells were screened. In96-well dishes, in 24-well dishes and in 25 cm² dishes, 120, 47 and 36hydridomas respectively were revealed to be secreting (revealed by IIF).

Cloning

The characteristic secreting hybridomas were cloned by limited dilution,4 clones were the subject of a particular study (B3, C7, D9 and F5).

The results obtained are given in Table 13.

TABLE 13 Strains/ascites IB5/B3 IIAIC7 IVD4/D9 IVD6/F5 L. infantumpromastigotes − ± − + L. amazonensis promastigotes ++({fraction (1/10)})+({fraction (1/100)}) +({fraction (1/1000)}) +++({fraction (1/1000)}) L.amazonensis amastigotes + − + + T. cruzi +/− − − − Molecular targetsunder 32 and 50 kDa 32 and 50 kDa between 30 and 36 kDa 60 kDa andimmunoprecipitation or Blotting 45 kDa

EXAMPLE 20 Obtaining Amastigote Forms Resistant to a Medicament

Promastigote forms of L. mexicana resistant to pentamidine are obtainedby medicamentous pressure in vitro and have a resistance index of about30. They are then transformed according to the culture method alreadydescribed into corresponding cultured amastigote forms which, afterdetermination of the IC50, retain the resistance to pentamidine inducedin the promastigote forms.

FIG. 13 gives the variation in the percentage of growth relative to acontrol without drugs as a function of the concentration of pentamidinein μmole/l (curve corresponds to the untreated promastigote forms of L.mexicana, curve corresponds to the resistant promastigote forms andcurve corresponds to the resistant amastigote forms. (IC50:concentration of pentamidine for which a growth inhibition of 50% isobserved): IC50 untreated 1.2±0.05 μM, IC50 resistant amastigote 32±1 μMand IC50 resistant promastigote 33±1 μM. The resistance index is equalto IC50 of the resistant strain/IC50 of the untreated strain. For thepromastigotes the value is 27 and for the amastigotes it is 26.

Due to the production of the development cycle in vitro, the inventionproduces amastigote forms of leishmania which are resistant to amedicament. This new experimental model allows the mechanism(s) involvedin the induction of chemical resistance in the parasitic stage presentin the infected host to be very closely studied.

What is claimed is:
 1. A method for the in vitro culture of differentstages of tissue parasites, consisting of culturing said parasites in atotally defined culture medium, which is an axenic, monophasic liquidculture medium, which is devoid of serum, and for obtaining amastigoteforms, is buffered at a pH of 5.5 to 6.5 and has an osmolarity of atleast 400 milliosmoles/kg of liquid, or, for obtaining promastigoteforms, is buffered at a pH of 7 to 7.5 and has an osmolarity of at least300 milliosmoles/kg of liquid, wherein said culture medium contains abasic culture medium for insect cells and at least one of an inorganicsalt, a source of amino acids, and a sugar.
 2. The method of claim 1,wherein the culture medium for insect cells is 199H M medium and theinorganic salts are Hanks' salts.
 3. The method of claim 1, wherein theproducts which are sources of amino acids are selected from the groupconsisting of L-glutamine and soja bean extracts.
 4. The method of claim1, wherein the sugars are D-glucose.
 5. The method of claim 1,comprising the steps of adding to the base medium, at the time of use,an anti-oxidizing agent, an agent with a reducing effect and vitamins.6. The method of claim 5, wherein, for the culture of amastigote forms,said culture medium contains a base medium consisting essentially of 8to 15% (v/v) of said cellular culture medium, 4 to 8% (w/v) of aminoacids, 2 to 4% (w/v) of sugars, 0.0002 to 0.0015 (w/v) of anti-oxidizingagent, 0.05% (w/v) of reducing agent, and 1 to 5% (v/v) of vitamins. 7.The method of claim 6, wherein, for the culture of amastigote forms,said culture medium contains a base medium consisting essentially of 10%(v/v) of said cellular culture medium, 5 to 6% (w/v) of amino acids, 2to 3% (w/v) of sugars, 0.0005% (w/v) of anti-oxidizing agent, 0.025%(w/v) of reducing agent, and 2% (v/v) of vitamins.
 8. The method ofclaim 1, wherein the culture medium for insect cells further comprisesat least one compound selected from the group consisting of a sulphurouscompound, an anti-oxidizing agent, a reducing agent, and a vitamin. 9.The method of claim 8, wherein said at least one compound is selectedfrom the group consisting of a sulphurous compound which is present inan amount of 0.25 to 0.50% (w/v) and bathocuproine sulphonic acid whichis present in an amount of 0.004 to 0.008% (v/v).
 10. The method ofclaim 9, wherein said sulphurous compound and bathocuproine sulphonicacid are present in an amount of 0.3% (w/v) and 0.005% (v/v)respectively.
 11. The method of claim 8, wherein said sulphurouscompound is cysteine.
 12. The method of claim 1, wherein culturing ofpromastigote stages is carried out in a culture medium comprising RPMI1640 medium, amino-acids and a buffer to adjust the pH to a value of 7to 7.5, and 199H M medium containing inorganic salts and anti-oxidizingagents.
 13. The method of claim 12, wherein the inorganic salts areHank's salts and the anti-oxidizing agent is hemin.
 14. The method ofclaim 12, comprising using at least 2% (v/v) of 199H M medium and 0.0002to 0.0015% (w/v) of anti-oxidizing agent.
 15. The method of claim 14,comprising using 2 to 10% (v/v) of 199H M medium, and 0.0005% ofanti-oxidizing agent.
 16. The method of claim 1, wherein the tissueparasites, are selected from the group consisting of Leishmania. T.cruzi and hemoprotozoa.
 17. A method for the in vitro culture ofdifferent stages of tissue parasites, consisting of culturing saidparasites in a totally defined culture medium, which is an axenic,monophasic liquid culture medium, which is devoid of serummacromolecules which are non-dialyzable at a cut-off threshold of 3 kDa,and for obtaining amastigote forms, is buffered at a pH of 5.5 to 6.5and has an osmolarity of at least 400 milliosmoles/kg of liquid, or, forobtaining promastigote forms, is buffered at a pH of 7 to 7.5 and has anosmolarity of at least 300 milliosmoles/kg of liquid, wherein saidculture medium contains a basic culture medium for insect cellsincluding at least one of an inorganic salt, a source of amino acids,and a sugar.